Materials and methods for detection of nucleic acids

ABSTRACT

Assays using non-natural bases are described. In one embodiment, the method involves contacting a sample suspected of containing the target nucleic acid with a polymerase and first and second primers; amplifying the target nucleic acid by PCR using the first and second primers to generate an amplification product having a double-stranded region and a single-stranded region that comprises the non-natural base; contacting the sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; annealing at least a portion of the reporter to the single-stranded region of the amplification product; and correlating a signal of the label with the presence of the target nucleic acid in the sample. The invention also provides corresponding kits for use in detecting target nucleic acids in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of co-pending U.S. applicationSer. No. 12/433,991, filed May 1, 2009, which is a continuation of Ser.No. 11/546,631, filed Oct. 12, 2006, now U.S. Pat. No. 7,541,147, whichis a continuation of Ser. No. 09/861,292, filed on May 18, 2001, nowU.S. Pat. No. 7,422,850, which claims priority to U.S. ProvisionalApplication Nos. 60/282,831, filed on Apr. 10, 2001; 60/240,398, filedon Oct. 14, 2000; and 60/205,712, filed on May 19, 2000. Theaforementioned applications are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

Polymerase chain reaction (PCR) is a method for the enzymaticamplification of specific segments of DNA. The PCR is based on repeatedcycles of the following basic steps: denaturation of double-strandedDNA, followed by oligonucleotide primer annealing to the DNA template,and primer extension by a nucleic acid polymerase (Mullis et al. andSaiki et al. 1985; and U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159, the entire disclosures of which are incorporated herein byreference). The oligonucleotide primers used in PCR are designed toanneal to opposite strands of the DNA, and are positioned so that thenucleic acid polymerase-catalyzed extension product of one primer canserve as the template strand for the other primer. The PCR amplificationprocess results in the exponential increase of discrete DNA fragmentswhose length is defined by the 5′ ends of the oligonucleotide primers.

While the PCR technique as presently practiced is an extremely powerfulmethod for amplifying nucleic acid sequences, the detection of theamplified material typically requires additional manipulation andsubsequent handling of the PCR products to determine whether the targetDNA is present. It is desirable to develop new methods and assays.

U.S. Pat. No. 5,210,015, incorporated herein by reference, teaches amethod for detecting a target nucleic acid using labeledoligonucleotides. The process uses a polymerase with 5′ to 3′ nucleaseactivity to cleave annealed labeled oligonucleotide probe which can thenbe detected.

U.S. Pat. No. 5,846,717, incorporated herein by reference, teaches amethod for detection of a target nucleic acid by forming a nucleic acidcleavage structure on the target sequence and then cleaving the nucleicacid cleavage structure in a site-specific manner using an enzyme with5′ nuclease activity.

U.S. Pat. No. 5,432,272, incorporated herein by reference, disclosesnon-standard bases that base pair in DNA or RNA but with a hydrogenbonding pattern different from the pattern observed with standard A:T orG:C base pairs.

SUMMARY OF THE INVENTION

The subject invention concerns materials and methods for the rapiddetection of a target nucleic acid. Methods of the invention employ areporter oligonucleotide; a nucleic acid polymerase; and first andsecond oligonucleotide primers, where at least one of the first andsecond oligonucleotide primers contains at least one non-natural base.

In one embodiment, the invention provides a method for detecting atarget nucleic acid in a sample, the method comprising contacting thesample with a nucleic acid polymerase, a first oligonucleotide primercomprising a sequence complementary to a first portion of the targetnucleic acid, a second oligonucleotide primer comprising a first regionand a second region, the first region comprising a sequencecomplementary to a second portion of the target nucleic acid and thesecond region comprising a non-natural base; amplifying the targetnucleic acid, if present in the sample, by PCR using the first andsecond oligonucleotide primers to generate an amplification producthaving (i) a double-stranded region and (ii) a single-stranded regionthat comprises the non-natural base; contacting the sample with areporter comprising a label and a non-natural base that is complementaryto the non-natural base of the single-stranded region; annealing atleast a portion of the reporter to the single-stranded region of theamplification product; cleaving, after annealing, at least a portion ofthe reporter to release at least one reporter fragment; and correlatingthe release of the at least one reporter fragment with the presence ofthe target nucleic acid in the sample.

In another embodiment, the invention provides a method for detecting atarget nucleic acid in a sample, the method comprising contacting thesample with a nucleic acid polymerase, a first oligonucleotide primercomprising a sequence complementary to a first portion of the targetnucleic acid, a second oligonucleotide primer comprising a first regionand a second region, the first region comprising a sequencecomplementary to a second portion of the target nucleic acid and thesecond region comprising a non-natural base; amplifying the targetnucleic acid, if present in the sample, by PCR using the first andsecond oligonucleotide primers to generate an amplification producthaving (i) a double-stranded region and (ii) a single-stranded regionthat comprises the non-natural base; contacting the sample with areporter comprising a label and a non-natural base that is complementaryto the non-natural base of the single-stranded region; incorporating thereporter into the amplification product opposite the non-natural base ofthe single-stranded region; and correlating the incorporating of thereporter with the presence of the target nucleic acid in the sample.

In yet another embodiment, the invention provides kits for detection oftarget nucleic acid. In one embodiment, the kit comprises a nucleic acidpolymerase; a first oligonucleotide primer comprising a sequencecomplementary to a first portion of the target nucleic acid; a secondoligonucleotide primer comprising a first region and a second region,the first region comprising a sequence complementary to a second portionof the target nucleic acid and the second region comprising anon-natural base; and a reporter comprising a label and a non-naturalbase that is complementary to the non-natural base of thesingle-stranded region. Optionally, the kit further comprises othercomponents such as buffers and reagents to perform the methods of theinvention.

The methods of the invention can be incorporated into a variety of massscreening techniques and readout platforms.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thefollowing detailed description of various embodiments of the inventionin connection with the accompanying drawings, in which:

FIG. 1A-1E schematically illustrate an assay method according to oneembodiment of the invention;

FIG. 2A-2E schematically illustrate an assay method according to asecond embodiment of the invention;

FIG. 3 displays chemical structures for a number of non-natural bases,where A is the point of attachment to a polymeric backbone, X is N orC—Z, Y is N or C—H, and Z is H, a substituted or unsubstituted alkylgroup, or a halogen;

FIG. 4A-4D schematically illustrate an assay method according to a thirdembodiment of the invention;

FIG. 5A-5E schematically illustrate an assay method according to afourth embodiment of the invention;

FIG. 6A-6E schematically illustrate an assay method according to a fifthembodiment of the invention;

FIG. 7A-7E schematically illustrate an assay method according to a sixthembodiment of the invention;

FIG. 8A-8E schematically illustrate an assay method according to aseventh embodiment of the invention;

FIG. 9A-9E schematically illustrate an assay method according to aneighth embodiment of the invention;

FIG. 10A-10E schematically illustrate an assay method according to aninth embodiment of the invention;

FIG. 11A-11E schematically illustrate an assay method according to atenth embodiment of the invention;

FIG. 12A-12E schematically illustrate an assay method according to aneleventh embodiment of the invention;

FIG. 13 schematically illustrates a general procedure for preparing anassay plate that contains allele specific PCR mixtures and templatesamples;

FIG. 14 is a graph demonstrating quenching of fluorescence in a PCRreaction by site specific incorporation of a quenching compound into aPCR amplification product, relative fluorescence units (RFU's) areindicated on the Y axis and the number of PCR cycles are indicated onthe X axis;

FIG. 15 schematically illustrates a synthesis scheme for the preparationof labeled non-natural bases according to Process A;

FIG. 16 schematically illustrates a synthesis scheme for the preparationof labeled non-natural bases according to Process B;

FIG. 17A is a graph demonstrating the “real time” monitoring ofquenching of fluorescence in a PCR reaction by site specificincorporation of a quenching compound into a PCR amplification product;relative fluorescence units (RFU's) are indicated on the Y axis and thenumber of PCR cycles are indicated on the X axis, FIG. 17B is a graphdemonstrating a melting curve analysis of the PCR products of FIG. 17A;the melting temperature is indicated on the X axis;

FIG. 18A is a graph demonstrating the “real time” monitoring ofquenching of fluorescence in a PCR reaction by site specificincorporation of a quenching compound into a PCR amplification product;relative fluorescence units (RFU's) are indicated on the Y axis and thenumber of PCR cycles are indicated on the X axis, FIG. 18B is a graphdemonstrating a melting curve analysis of the PCR products of FIG. 18A;the melting temperature is indicated on the X axis;

FIG. 19 is a graph demonstrating the “real time” monitoring of anincrease in the fluorescence in a PCR reaction amplifying genomic DNA;relative fluorescence units (RFU's) are indicated on the Y axis and thenumber of PCR cycles are indicated on the X axis;

FIG. 20 is a graph demonstrating the “real time” monitoring of anincrease in the fluorescence in PCR reaction amplifying differentamounts of reverse-transcribed RNA; relative fluorescence units (RFU's)are indicated on the Y axis and the number of PCR cycles are indicatedon the X axis;

FIG. 21 is a graph demonstrating the “real time” monitoring of quenchingof fluorescence in a PCR reaction amplifying different amounts ofreverse-transcribed RNA by site specific incorporation of a quenchingcompound into PCR amplification products; relative fluorescence units(RFU's) are indicated on the Y axis and the number of PCR cycles areindicated on the X axis;

FIG. 22 is a graph demonstrating the combined results from the multiplexPCR analysis of wild type, mutant, and heterozygous Factor V DNAtargets; HEX fluorescence RFUs are shown on the Y axis and FAMfluorescence RFUs are shown on the X axis;

FIG. 23A-B are graphs demonstrating the combined results from themultiplex PCR analysis of polymorphisms in mouse STS sequence 27MMHAP25FLA6 of genomic DNA from various mouse strains; PCR cycle numberis indicated on the X axis. In FIG. 23A HEX fluorescence RFUs are shownon the Y axis; in FIG. 23B FAM fluorescence RFUs are shown on the Yaxis; and

FIG. 24 is a melt curve analysis of the PCR products from a reactionamplifying different amounts of reverse-transcribed RNA by site specificincorporation of a quenching compounds; the change in fluorescence overtime is indicated on the Y axis and melting temperature is indicated onthe X axis.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns methods and materials for detecting,analyzing mutations in, or quantifying the amount of a target nucleicacid in a sample. Methods of the invention generally include the use ofPCR. The PCR can be a Fast-Shot™ amplification. The methods of thepresent invention employ a reporter oligonucleotide; a nucleic acidpolymerase; and first and second oligonucleotide primers, where at leastone of the first and second primer oligonucleotides contains at leastone non-natural base. Other related assay methods for use with solidsupports are described in U.S. Patent Provisional Application Ser. No.60/240,398, entitled “Solid Support Assay Systems and Methods UtilizingNon-natural Bases,” filed Oct. 14, 2000.

As used herein, “nucleic acids” include polymeric molecules such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleicacid (PNA), or any sequence of what are commonly referred to as basesjoined by a chemical backbone where the bases have the ability to formbase pairs or hybridize with a complementary chemical structure.Suitable non-nucleotidic backbones include, for example, polyamide andpolymorpholino backbones. The term “nucleic acids” includesoligonucleotide, nucleotide, or polynucleotide sequences, and fragmentsor portions thereof. The nucleic acid can be provided in any suitableform, e.g., isolated from natural sources, recombinantly produced, orartificially synthesized, can be single- or double-stranded, and canrepresent the sense or antisense strand.

The term “oligonucleotide” refers generally to short chain (e.g., lessthan about 100 nucleotides in length, and typically about 6 to about 50nucleotides in length) nucleic acids that can be prepared usingtechniques presently available in the art such as, for example, solidsupport nucleic acid synthesis, DNA replication, reverse transcription,restriction digest, run-off transcription, or the like. The exact sizeof the oligonucleotide will depend upon many factors, which in turn willdepend upon the ultimate function or use of the oligonucleotide.

A “sequence” refers to an ordered arrangement of nucleotides.

The term “sample” is used in its broadest sense. The term includes aspecimen or culture (e.g., microbiological cultures), as well asbiological and non-biological samples.

As used herein, “target” or “target nucleic acid” refers to a nucleicacid containing a nucleic acid sequence, suspected to be in a sample andto be detected or quantified in the method or system of the invention.Target nucleic acids contain the target nucleic acid sequences that areactually assayed during an assay procedure. The target can be directlyor indirectly assayed. In at least some embodiments, the target nucleicacid, if present in the sample, is used as a template for amplificationaccording to the methods of the invention.

As used herein, the terms “complementary” or “complementarity,” whenused in reference to nucleic acids (i.e., a sequence of nucleotides suchas an oligonucleotide or a target nucleic acid), refer to sequences thatare related by base-pairing rules. For natural bases, the base pairingrules are those developed by Watson and Crick. For non-natural bases, asdescribed herein, the base-pairing rules include the formation ofhydrogen bonds in a manner similar to the Watson-Crick base pairingrules or by hydrophobic, entropic, or van der Waals forces. As anexample, for the sequence “T-G-A”, the complementary sequence is“A-C-T.” Complementarity can be “partial,” in which only some of thebases of the nucleic acids are matched according to the base pairingrules. Alternatively, there can be “complete” or “total” complementaritybetween the nucleic acids. The degree of complementarity between thenucleic acid strands has effects on the efficiency and strength ofhybridization between the nucleic acid strands.

The term “hybridization” is used in reference to the pairing ofcomplementary nucleic acids. Hybridization and the strength ofhybridization (i.e., the strength of the association between the nucleicacids) is influenced by such factors as the degree of complementaritybetween the nucleic acids, stringency of the hybridization conditionsinvolved, the melting temperature (T_(m)) of the formed hybrid, and theG:C ratio within the nucleic acids.

As used herein, “label” refers to any atom or molecule which can providea detectable (preferably quantifiable) signal, and which can be attachedto a nucleic acid or protein. Labels can provide signals detectable bysuch techniques as colorimetric, fluorescent, electrophoretic,electrochemical, spectroscopic, chromatographic, densitometric, orradiographic techniques, and the like. Labels can be molecules that donot themselves produce a detectable signal, but when used in conjunctionwith another label can produce or quench a detectable signal. Forexample, a label can be a quencher of a quencher-dye pair.

As used herein, the term “thermostable nucleic acid polymerase” refersto an enzyme that catalyzes the polymerization of nucleosides and whichis relatively stable to heat when compared, for example, to nucleotidepolymerases from E. coli. Generally, the enzyme will initiate synthesisat the 3′-end of the primer annealed to the target sequence, and willproceed in the 5′-direction along the template, and if possessing a 5′to 3′ nuclease activity, hydrolyzing an intervening, annealedoligonucleotide to release intervening nucleotide bases oroligonucleotide fragments, until synthesis terminates. A thermostableenzyme has activity at a temperature of at least about 37° C. to about42° C., typically in the range from about 50° C. to about 75° C.Representative thermostable polymerases include, for example,thermostable polymerases such as native and altered polymerases ofThermus species, including, but not limited to, Thermus aquaticus (Taq),Thermus flavus (Tfl), and Thermus thermophilus (Tth), and of theThermotoga species, including, but not limited to, Thermotoganeapolitana.

As used herein, the term “DNA polymorphism” refers to the condition inwhich two or more different nucleotide sequences can exist at aparticular site in DNA and includes any nucleotide variation, such assingle or multiple nucleotide substitutions, deletions or insertions.These nucleotide variations can be mutant or polymorphic allelevariations. At least some of the embodiments of the methods describedherein can detect single nucleotide changes in nucleic acids such asoccur in β-globin genetic diseases caused by single-base mutations,additions or deletions (some β-thalassemias, sickle cell anemia,hemoglobin C disease, etc.), as well as multiple-base variations such asare involved with α-thalassemia or some β-thalassemias. In addition, theprocess herein can detect polymorphisms, which are not necessarilyassociated with a disease, but are merely a condition in which two ormore different nucleotide sequences (whether having substituted, deletedor inserted nucleotide base pairs) can exist at a particular site in thenucleic acid in the population, as with HLA regions of the human genomeand random polymorphisms such as mitochondrial DNA.

The present invention provides methods and materials for detecting atarget nucleic acid in a sample. In one embodiment, a method includescontacting a sample suspected of containing the target nucleic acid witha polymerase and first and second primers; amplifying the target nucleicacid, if present in the sample, by PCR using the first and secondprimers to generate an amplification product having a double-strandedregion and a single-stranded region that comprises at least onenon-natural base; contacting the sample with a reporter comprising alabel and a non-natural base (or bases) that is complementary to thenon-natural base (or bases) of the single-stranded region; annealing atleast a portion of the reporter to the single-stranded region of theamplification product; cleaving, after annealing, at least a portion ofthe reporter to release at least one reporter fragment; and correlatingthe release of the at least one reporter fragment with the presence ofthe target nucleic acid in the sample.

In another embodiment, a method includes contacting a sample suspectedof containing the target nucleic acid with a polymerase and first andsecond primers; amplifying the target nucleic acid, if present in thesample, by PCR using the first and second primers to generate anamplification product having a double-stranded region and asingle-stranded region that comprises at least one non-natural base;contacting the sample with a reporter comprising a label and anon-natural base (or bases) that is complementary to the non-naturalbase (or bases) of the single-stranded region; incorporating thereporter into the amplification product; and correlating theincorporating of the reporter with the presence of the target nucleicacid in the sample.

The invention also includes corresponding kits for use in detectingtarget nucleic acids in a sample using one or more of the methodsdescribed herein.

The invention can provide a number of advantages, if desired, including,in some embodiments, the ability to detect target nucleic acid in asample without the need for post-reaction processing such as washing orseparation (e.g., by gel electrophoresis). In addition, in someembodiments, the method can be performed by adding all of the elementsinto one reaction mixture that is processed using one set of reactionconditions. This can, in turn, avoid or reduce problems or concernsassociated with multiple reaction steps and reagents.

General Discussion

One embodiment of the invention will now be described in general termswith reference to the schematic representation shown in FIG. 1.Referring to FIG. 1A, a sample is suspected to contain target nucleicacid 100, the target nucleic acid 100 including a first portion 102 anda second portion 104. As shown, target nucleic acid 100 is adouble-stranded molecule comprised of strands 100 a and 100 b.

Referring to FIG. 1B, the sample is contacted with a first primer 106and a second primer 108 as illustrated. The first primer 106 iscomplementary to the first portion 102 of the target nucleic acid 100.The second primer 108 includes a first region 110 and a second region112, the first region 110 comprising a sequence that is complementary tothe second portion 104 of the target nucleic acid 100. The second region112 of the second primer 108 includes a non-natural base 114. The secondregion 112 is not complementary to the target nucleic acid 100.

In addition to the first primer and the second primer, the sample isalso contacted with a polymerase and subjected to polymerase chainreaction (PCR), as herein described. If the target nucleic acid 100 ispresent in the sample, the complementary region of the first primer 106and the complementary region of the second primer 108 anneal to thecorresponding portions 102 and 104 of the target nucleic acid 100following standard base-pairing rules. As shown, when the primers areannealed to the target, the 3′ terminal nucleotide of the first primer106 is separated from the 3′ terminal nucleotide of the second primer108 by a sequence of nucleotides, or a “gap,” depicted as 107 in FIG.1B. In a preferred embodiment, the first and second oligonucleotideprimers are designed such that a gap 107 of between about zero (0) toabout five (5) bases on the template nucleic acid exists between the 3′ends of the PCR primers when annealed to the template nucleic acid.

As shown in FIG. 1B, the polymerase is used to synthesize a singlestrand from the 3′-OH end of each primer, using PCR, or Fast-Shot™amplification. That is, first primer 106 is used to synthesize strand120 a that is complementary to at least a portion of strand 100 a of thetarget nucleic acid 100, and the second primer 108 is used to synthesizestrand 120 b that is complementary to at least a portion of strand 100 bof the target nucleic acid 100, as illustrated in FIG. 1C. Thepolymerase chain reaction is allowed to proceed for the desired numberof cycles to obtain an amplification product 120.

As shown in FIG. 1C, the amplification product 120 includes adouble-stranded region 122 and a single-stranded region 124. As shown inthis embodiment, the non-natural base 114 is located in thesingle-stranded region 124, adjacent the double-stranded region 122. Thesingle-stranded region 124 can include more than one non-natural base.

Referring now to FIG. 1D, the amplification product 120 is contactedwith a reporter 126. It is contemplated that the reporter 126 can beadded to the reaction before, during, or after, amplification of thetarget nucleic acid has occurred. The reporter 126 comprises a label128, 132 and a non-natural base 130 that is complementary to thenon-natural base 114 of the single-stranded region 124 of theamplification product 120. In the embodiment shown in FIG. 1D, thereporter 126 includes a label comprising a dye 128 and a quencher 132,and a non-natural base 130. The reporter 126 is allowed to anneal to theamplification product 120. After annealing, at least a portion of thereporter 126 is cleaved, generating a reporter fragment 134 thatincludes dye 128, as illustrated in FIG. 1E. The release of the reporterfragment 134 is correlated with the presence of the target nucleic acidin the sample. In the illustrated case, the presence of the unquencheddye of the reporter fragment is detected. In an alternative embodiment,the positions of the dye and quencher are reversed, with the reporterfragment carrying away the quencher upon cleavage.

FIG. 8 illustrates an alternative assay in which the quencher 132 iscoupled to the second primer 108 instead of the reporter. The quencher132 quenches the fluorescence of the dye 128 of the reporter 126 until aportion of the reporter 126 is cleaved to generate a reporter fragment134 that includes the dye 128. As an alternative, the quencher can becoupled to the reporter and the dye can be coupled to the second primer.In the present description, elements in common between the embodimentsof the figures are numbered identically, and such elements need not beseparately discussed.

FIGS. 9A-9E, 10A-10E, 11A-11E, and 12A-12E illustrate a number ofembodiments similar to the assay of FIGS. 1A-1E where X and Y representnon-standard bases. For example, X can represent iso-cytidine (iso-C)and Y can represent iso-guanosine (iso-G). The following descriptionsillustrate the differences between the assay of FIGS. 1A-1E and theseembodiments. Otherwise, the same considerations and conditions areapplicable.

In the assay of FIGS. 9A-9E, the first and second primers 106, 108 arebrought into contact with the double stranded target nucleic acid 100,as illustrated in FIG. 9A. The second primer has a first region 110 thatis complementary to a portion of the target nucleic acid and a secondregion 112 that is not complementary to the target nucleic acid and doesnot typically hybridize to the target nucleic acid. The second primer108 has a non-standard base 114 in the second region 112 and adjacent tothe first region 110 of the second primer that anneals to the targetnucleic acid. The first and second primers are used to synthesize by PCRan amplification product 120 that is complementary to portions of thetarget nucleic acid, as illustrated in FIGS. 9B and 9C. Theamplification product 120 has a double stranded region 122 and a singlestranded region 124. A reporter 126 is brought into contact with thesingle stranded region 124 of the amplification product 120, asillustrated in FIG. 9C. The reporter includes a non-standard base 119that is complementary to the non-standard base 114 of the singlestranded region 124. The reporter 126 anneals to the single strandedregion 124, as illustrated in FIG. 9D. In the reporter 126, a base 127adjacent the non-standard base 119 can be complementary, but is notnecessarily so, to the base 131 of the double-stranded region adjacentto the non-standard base 114 of the single stranded region. The base 127is cleaved by the polymerase to form a reporter fragment 134, asillustrated in FIG. 9E, that typically contains a label or a portion 128of a label, such as a fluorophore or quencher to allow the detection ofthe reporter fragment of the amplification product 120. Optionally, base127 is replaced with an oligonucleotide sequence that typically includesthe label 128 and is cleaved from the remainder of the reporter.

Another embodiment is illustrated in FIGS. 10A-10E. In this embodiment,base 131 is not part of the first region 110 of the second primer 108that is complementary to the target nucleic acid sequence, but insteadbase 131 is non-complementary to the target nucleic acid sequence and ispart of the second region 112 of the second primer 108. Otherwise, thesteps and procedures of this assay are the same as those of the assay ofFIGS. 9A-9E.

In another embodiment, the first and second primers 106, 108 are broughtinto contact with the double stranded target nucleic acid 100, asillustrated in FIG. 11A. The second primer has a first region 110 thatis complementary to a portion of the target nucleic acid and a secondregion 112 that is not complementary to the target nucleic acid and doesnot typically hybridize to the target nucleic acid. The second primer108 has at least two consecutive non-standard bases 114, 117 in thesecond region 112 and adjacent to the first region 110 of the secondprimer that anneals to the target nucleic acid. The first and secondprimers are used to synthesize by PCR an amplification product 120 thatis complementary to portions of the target nucleic acid, as illustratedin FIGS. 11B and 11C. The amplification product 120 has a doublestranded region 122 and a single stranded region 124. Optionally, a base141 is misincorporated across from the first of the non-standard bases114, 117. A reporter 126 is brought into contact with the singlestranded region 124 of the amplification product 120, as illustrated inFIG. 11C. The reporter includes non-standard bases that arecomplementary to the non-standard bases of the second primer. Thereporter 126 anneals to the single stranded region 124, as illustratedin FIG. 11D. Non-standard base 127 is cleaved by the polymerase to forma reporter fragment 134, as illustrated in FIG. 11E, that typicallycontains a label or a portion 128 of a label, such as a fluorophore orquencher to allow detection of the reporter fragment or theamplification product 120. Optionally, base 127 is replaced with anoligonucleotide sequence that typically includes the label 128 and iscleaved from the remainder of the reporter.

In yet another embodiment, the first and second primers 106, 108 arebrought into contact with the double stranded target nucleic acid 100,as illustrated in FIG. 12A. The second primer has a first region 110that is complementary to a portion of the target nucleic acid and asecond region 112 that is not complementary to the target nucleic acidand does not typically hybridize to the target nucleic acid. The secondprimer 108 has two non-standard bases 114, 117 in the second region 112and adjacent to the first region 110 of the second primer that annealsto the target nucleic acid. The first and second primers are used tosynthesize by PCR an amplification product 120 that is complementary toportions of the target nucleic acid, as illustrated in FIGS. 12B and12C. The amplification product 120 has a double stranded region 122 anda single stranded region 124. Optionally, the amplification product 120includes a base 121 misincorporated by the polymerase across fromnon-standard base 117. A reporter 126 is brought into contact with thesingle stranded region 124 of the amplification product 120, asillustrated in FIG. 12C. The reporter includes a non-standard base thatis complementary to the non-standard base 114 of the second primer. Thereporter 126 anneals to the single stranded region 124, as illustratedin FIG. 12D. The reporter 126 includes a base 127 coupled to anon-standard base 119 that anneals to base 114 of the single-strandedregion, but the base 127 is not complementary to the base 117 of thesingle-stranded region. Base 127 is cleaved by the polymerase to form areporter fragment 134, as illustrated in FIG. 12E, that typicallycontains a label or a portion 128 of a label, such as a fluorophore orquencher, to allow detection of the reporter fragment or theamplification product 120. Optionally, base 127 is replaced with anoligonucleotide sequence that typically includes the label 128 and iscleaved from the remainder of the reporter.

Another embodiment of the invention is shown schematically in FIG. 2. Asshown in FIG. 2A, a double-stranded target nucleic acid 100 includes afirst portion 102 and a second portion 104. The sample is contacted witha first primer 106 and a second primer 108. The first primer 106 iscomplementary to the first portion 102 of the target nucleic acid 100.The second primer 108 includes a first region 110 that is complementaryto the second portion 104 of the target nucleic acid 100, and a secondregion 114 that comprises a non-natural base 114 and is notcomplementary to the target nucleic acid 100.

In addition to the first primer 106 and second primer 108, the sample iscontacted with a polymerase (not shown), and a polymerase chain reactionis run. Similar to the embodiment shown in FIG. 1, if the target nucleicacid 100 is present in the sample, the complementary portion of thefirst primer 106 and the complementary portion 110 of the second primer108 will anneal to the corresponding regions 102, 104 of the targetnucleic acid 100 following standard base-pairing rules. Similar to theembodiment shown in FIG. 1, when the primers are annealed to the target,the 3′ terminal nucleotide of the first primer 106 is separated from the3′ terminal nucleotide of the second primer 108 by a sequence ofnucleotides, or a “gap” 107. In a preferred embodiment, the first andsecond oligonucleotide primers are designed such that a gap of betweenabout zero (0) to about five (5) bases on the template nucleic acidexists between the 3′ ends of the PCR primers when annealed to thetemplate nucleic acid.

As shown in FIGS. 2B and 2C, the polymerase is used to synthesize asingle strand 120 a, 120 b from the 3′-OH end of each primer, usingpolymerase chain reaction, or a Fast-Shot™ amplification. The polymerasechain reaction is allowed to proceed for the desired number of cycles,to obtain an amplification product 120 shown in FIG. 2C.

As shown in FIG. 2C, the amplification product 120 includes adouble-stranded region 122 and a single-stranded region 124. As shown,the single-stranded region 124 comprises the non-natural base 114 of thesecond primer 108. Although the single-stranded region 124 is shownincluding a single non-natural base, the invention is not so limited,and the single-stranded region can include more than one non-naturalbase.

Referring now to FIG. 2D, the amplification product 120 is contactedwith a reporter 150. The reporter 150 is added to the sample before,during or after PCR amplification. The reporter 150 comprises a label154 and a non-natural base 152 that is complementary to the non-naturalbase 114 of the single-stranded region 124 of the amplification product120, as illustrated in FIG. 2E. The reporter 150 is incorporated intothe amplification product opposite the non-natural base 114. Asdiscussed in more detail below, incorporation of the reporter 150 can beaccomplished using any suitable enzyme, such as, for example, apolymerase or ligase. Presence of the target nucleic acid in the sampleis determined by correlating the presence of the reporter in theamplification product. In the illustrated case, for example, presence ofthe target nucleic acid is determined by detecting the label 154, forexample, by fluorescence or other visualization method. Suitabledetection and visualization methods will be described in more detailbelow.

While the schematic diagrams of FIGS. 1 and 2 show relative positionsand sizes of the components of the invention, these representations arefor illustrative purposes only. As will be apparent from the discussionherein, the relative sizes of the first primer and second primer, aswell as the first portion and second portion of the target nucleic acid,will vary depending upon the particular application. Further, therelative location of the first primer and the second primer along thetarget nucleic acid will vary. Additionally, the location of thenon-natural base and labels used in the invention will vary dependingupon application.

Polymerase

The invention provides methods and materials that utilize the polymerasechain reaction, or a Fast-Shot™ amplification, to detect a targetnucleic acid of interest in a sample. Suitable nucleic acid polymerasesinclude, for example, polymerases capable of extending anoligonucleotide by incorporating nucleic acids complementary to atemplate oligonucleotide. For example, the polymerase can be a DNApolymerase.

Enzymes having polymerase activity catalyze the formation of a bondbetween the 3′ hydroxyl group at the growing end of a nucleic acidprimer and the 5′ phosphate group of a nucleotide triphosphate. Thesenucleotide triphosphates are usually selected from deoxyadenosinetriphosphate (A), deoxythymidine triphosphate (T), deoxycytidinetriphosphate (C) and deoxyguanosine triphosphate (G). However, in atleast some embodiments, polymerases useful for methods of the presentinvention can also incorporate non-natural bases using nucleotidetriphosphates of those non-natural bases.

Because the relatively high temperatures necessary for stranddenaturation during methods such as PCR can result in the irreversibleinactivation of many nucleic acid polymerases, nucleic acid polymeraseenzymes useful for the invention preferably retain sufficient polymeraseactivity to complete the reaction when subjected to the temperatureextremes of methods such as PCR. Preferably, the nucleic acid polymeraseenzymes useful for methods of the invention are thermostable nucleicacid polymerases. Suitable thermostable nucleic acid polymerasesinclude, but are not limited to, enzymes derived from thermophilicorganisms. Examples of thermophilic organisms from which suitablethermostable nucleic acid polymerase can be derived include, but are notlimited to, Thermus aquaticus, Thermus thermophilus, Thermus flavus,Thermotoga neapolitana and species of the Bacillus, Thermococcus,Sulfobus, and Pyrococcus genera. Nucleic acid polymerases can bepurified directly from these thermophilic organisms. However,substantial increases in the yield of nucleic acid polymerase can beobtained by first cloning the gene encoding the enzyme in a multicopyexpression vector by recombinant DNA technology methods, inserting thevector into a host cell strain capable of expressing the enzyme,culturing the vector-containing host cells, then extracting the nucleicacid polymerase from a host cell strain which has expressed the enzyme.Suitable thermostable nucleic acid polymerases, such as those describedabove, are commercially available.

A number of nucleic acid polymerases possess activities in addition tonucleic acid polymerase activity; these can include 5′-3′ exonucleaseactivity and 3′-5′ exonuclease activity. The 5′-3′ and 3′-5′ exonucleaseactivities are known to those of ordinary skill in the art. The 3′-5′exonuclease activity improves the accuracy of the newly-synthesizedstrand by removing incorrect bases that have been incorporated. Incontrast, the 5′-3′ exonuclease activity often present in nucleic acidpolymerase enzymes can be undesirable in a particular application sinceit may digest nucleic acids, including primers that have an unprotected5′ end. Thus, a thermostable nucleic acid polymerase with an attenuated5′-3′ exonuclease activity, or in which such activity is absent, is adesired characteristic of an enzyme for use in at least some embodimentsof the invention. In other embodiments, the polymerase is desired tohave 5′-3′ exonuclease activity to efficiently cleave the reporter andrelease labeled fragments so that the signal is directly or indirectlygenerated.

Suitable nucleic acid polymerases having no 5′-3′ exonuclease activityor an attenuated 5′-3′ exonuclease activity are known in the art.Various nucleic acid polymerase enzymes have been described where amodification has been introduced in a nucleic acid polymerase whichaccomplishes this object. For example, the Klenow fragment of E. coliDNA polymerase I can be produced as a proteolytic fragment of theholoenzyme in which the domain of the protein controlling the 5′-3′exonuclease activity has been removed. Suitable nucleic acid polymerasesdeficient in 5′-3′ exonuclease activity are commercially available.Examples of commercially available polymerases that are deficient in5′-3′ exonuclease activity include AMPLITAQ STOFFEL™ DNA polymerase andKlenTaq™ DNA polymerase.

Polymerases can “misincorporate” bases during PCR. In other words, thepolymerase can incorporate a nucleotide (for example adenine) at the 3′position on the synthesized strand that does not form canonical hydrogenbase pairing with the paired nucleotide (for example, cytosine) on thetemplate nucleic acid strand. The PCR conditions can be altered todecrease the occurrence of misincorporation of bases. For example,reaction conditions such as temperature, salt concentration, pH,detergent concentration, type of metal, concentration of metal, and thelike can be altered to decrease the likelihood that polymerase willincorporate a base that is not complementary to the template strand.

As an alternative to using a single polymerase, any of the methodsdescribed herein can be performed using multiple enzymes. For example, apolymerase, such as an exo-nuclease deficient polymerase, and anexo-nuclease can be used in combination. Another example is the use ofan exo-nuclease deficient polymerase and a thermostable flapendonuclease. In addition, it will be recognized that RNA can be used asa sample and that a reverse transcriptase can be used to transcribe theRNA to cDNA. The transcription can occur prior to or during PCRamplification.

First Primer and Second Primer

The invention provides a method of detecting a target nucleic acid usingPCR that involves a polymerase, a first primer and a second primer. Asshown in FIGS. 1, 2, and 9-12, the first primer 106 comprises a sequencecomplementary to a first portion 102 of the target nucleic acid 100. Thesecond primer 108 comprises a first region 110 and a second region 112,the first region 110 comprising a sequence complementary to a secondportion 104 of the target nucleic acid and the second region 112comprising at least one non-natural base. The second region is generallynot complementary to the target nucleic acid.

In PCR techniques, the primers are designed to be complementary tosequences known to exist in a target nucleic acid to be amplified.Typically, the primers are chosen to be complementary to sequences thatflank (and can be part of) the target nucleic acid sequence to beamplified. Preferably, the primers are chosen to be complementary tosequences that flank the target nucleic acid to be detected. Once thesequence of the target nucleic acid is known, the sequence of a primeris prepared by first determining the length or size of the targetnucleic acid to be detected, determining appropriate flanking sequencesthat are near the 5′ and 3′ ends of the target nucleic acid sequence orclose to the 5′ and 3′ ends, and determining the complementary nucleicacid sequence to the flanking areas of the target nucleic acid sequenceusing standard Watson-Crick base pairing rules, and then synthesizingthe determined primer sequences. This preparation can be accomplishedusing any suitable methods known in the art, for example, cloning andrestriction of appropriate sequences and direct chemical synthesis.Chemical synthesis methods can include, for example, the phosphodiestermethod described by Narang et al., Methods in Enzymology, 68:90 (1979),the phosphodiester method disclosed by Brown et al., Methods inEnzymology, 68:109 (1979), the diethylphosphoramidate method disclosedin Beaucage et al., Tetrahedron Letters, 22:1859 (1981), and the solidsupport method disclosed in U.S. Pat. No. 4,458,066, all of which areincorporated herein by reference.

The ability of the first primer and second primer to form sufficientlystable hybrids to the target nucleic acid depends upon several factors,for example, the degree of complementarity exhibited between the primerand the target nucleic acid. Typically, an oligonucleotide having ahigher degree of complementarity to its target will form a more stablehybrid with the target.

Additionally, the length of the primer can affect the temperature atwhich the primer will hybridize to the target nucleic acid. Generally, alonger primer will form a sufficiently stable hybrid to the targetnucleic acid sequence at a higher temperature than will a shorterprimer.

Further, the presence of high proportion of G or C or of particularnon-natural bases in the primer can enhance the stability of a hybridformed between the primer and the target nucleic acid. This increasedstability can be due to, for example, the presence of three hydrogenbonds in a G-C interaction or other non-natural base pair interactioncompared to two hydrogen bonds in an A-T interaction.

Stability of a nucleic acid duplex can be estimated or represented bythe melting temperature, or “T_(m).” The T_(m) of a particular nucleicacid duplex under specified conditions is the temperature at which 50%of the population of the nucleic acid duplexes dissociate intosingle-stranded nucleic acid molecules. The T_(m) of a particularnucleic acid duplex can be predicted by any suitable method. Suitablemethods for determining the T_(m) of a particular nucleic acid duplexinclude, for example, software programs. Primers suitable for use in themethods and kits of the present invention can be predetermined based onthe predicted T_(m) of an oligonucleotide duplex that comprises theprimer.

As shown in FIGS. 1 and 2, when the first primer and second primer areannealed to the target nucleic acid, a gap 107 exists between the 3′terminal nucleotide of the first primer 106 and the 3′ terminalnucleotide of the second primer 108. The gap 107 comprises a number ofnucleotides of the target nucleic acid. The gap can be any number ofnucleotides provided that the polymerase can effectively incorporatenucleotides into an elongating strand to fill the gap during a round ofthe PCR reaction (e.g., a round of annealing, extension, denaturation).Typically, a polymerase can place about 30 to about 100 bases persecond. Thus, the maximum length of the gap between primers depends uponthe amount of time within a round of PCR where the temperature is in arange in which the polymerase is active and the primers are annealed.

For a Fast-Shot™ amplification, using a standard thermal cycler, thetemperature change is relatively slow given the limitations of thePeltier cooling and heating. When using a standard thermal cycler, thetime the Fast-Shot™ amplification reaction conditions are within atemperature range where the polymerase is active and the primer isannealed is about 10 to about 15 seconds. It is contemplated that themethods of the invention can be performed using a microfluidics systemcapable of rapidly thermal cycling the temperature of a sample, whereextension times are relatively short and temperature change isrelatively rapid. Such rapid thermal cycling can be performed using, forexample, LabChip™ technology (Caliper Technology, Palo Alto, Calif.). Inone embodiment, the first and second oligonucleotide primers aredesigned such that a gap of between about zero (0) to about five (5)bases on the target nucleic acid exists between the 3′ ends of the PCRprimers when annealed to the target nucleic acid.

Non-Natural Bases

As contemplated in the invention, the second region of the second primertypically comprises at least one non-natural base. DNA and RNA areoligonucleotides that include deoxyriboses or riboses, respectively,coupled by phosphodiester bonds. Each deoxyribose or ribose includes abase coupled to a sugar. The bases incorporated in naturally-occurringDNA and RNA are adenosine (A), guanosine (G), thymidine (T), cytidine(C), and uridine (U). These five bases are “natural bases”. According tothe rules of base pairing elaborated by Watson and Crick, the naturalbases can hybridize to form purine-pyrimidine base pairs, where G pairswith C and A pairs with T or U. These pairing rules facilitate specifichybridization of an oligonucleotide with a complementaryoligonucleotide.

The formation of these base pairs by the natural bases is facilitated bythe generation of two or three hydrogen bonds between the two bases ofeach base pair. Each of the bases includes two or three hydrogen bonddonor(s) and hydrogen bond acceptor(s). The hydrogen bonds of the basepair are each formed by the interaction of at least one hydrogen bonddonor on one base with a hydrogen bond acceptor on the other base.Hydrogen bond donors include, for example, heteroatoms (e.g., oxygen ornitrogen) that have at least one attached hydrogen. Hydrogen bondacceptors include, for example, heteroatoms (e.g., oxygen or nitrogen)that have a lone pair of electrons.

The natural bases, A, G, C, T, and U, can be derivatized by substitutionat non-hydrogen bonding sites to form modified natural bases. Forexample, a natural base can be derivatized for attachment to a supportby coupling a reactive functional group (for example, thiol, hydrazine,alcohol, amine, and the like) to a non-hydrogen bonding atom of thebase. Other possible substituents include, for example, biotin,digoxigenin, fluorescent groups, alkyl groups (e.g., methyl or ethyl),and the like.

Non-natural bases, alternatively referred to herein as non-standardbases, which form hydrogen-bonding base pairs, can also be constructedas described, for example, in U.S. Pat. Nos. 5,432,272, 5,965,364,6,001,983, and 6,037,120 and U.S. patent application Ser. No.08/775,401, all of which are incorporated herein by reference. FIG. 3illustrates several examples of suitable bases and their correspondingbase pairs. Specific examples of these bases include the following basesin base pair combinations (iso-C/iso-G, K/X, H/J, and M/N):

where A is the point of attachment to the sugar or other portion of thepolymeric backbone and R is H or a substituted or unsubstituted alkylgroup. It will be recognized that other non-natural bases utilizinghydrogen bonding can be prepared, as well as modifications of theabove-identified non-natural bases by incorporation of functional groupsat the non-hydrogen bonding atoms of the bases.

The hydrogen bonding of these non-natural base pairs is similar to thoseof the natural bases where two or three hydrogen bonds are formedbetween hydrogen bond acceptors and hydrogen bond donors of the pairingnon-natural bases. One of the differences between the natural bases andthese non-natural bases is the number and position of hydrogen bondacceptors and hydrogen bond donors. For example, cytosine can beconsidered a donor/acceptor/acceptor base with guanine being thecomplementary acceptor/donor/donor base. Iso-C is anacceptor/acceptor/donor base and iso-G is the complementarydonor/donor/acceptor base, as illustrated in U.S. Pat. No. 6,037,120,incorporated herein by reference.

Other non-natural bases for use in oligonucleotides include, forexample, naphthalene, phenanthrene, and pyrene derivatives as discussed,for example, in Ren et al., J. Am. Chem. Soc., 118:1671 (1996) andMcMinn et al., J. Am. Chem. Soc., 121:11585 (1999), both of which areincorporated herein by reference. These bases do not utilize hydrogenbonding for stabilization, but instead rely on hydrophobic or van derWaals interactions to form base pairs.

The use of non-natural bases according to the invention is extendablebeyond the detection and quantification of nucleic acid sequencespresent in a sample. For example, non-natural bases can be recognized bymany enzymes that catalyze reactions associated with nucleic acids.While a polymerase requires a complementary nucleotide to continuepolymerizing an extending oligonucleotide chain, other enzymes do notrequire a complementary nucleotide. If a non-natural base is present inthe template and its complementary non-natural base is not present inthe reaction mix, a polymerase will typically stall (or, in someinstances, misincorporate a base when given a sufficient amount of time)when attempting to extend an elongating primer past the non-naturalbase. However, other enzymes that catalyze reactions associated withnucleic acids, such as ligases, kinases, nucleases, polymerases,topoisomerases, helicases, and the like can catalyze reactions involvingnon-natural bases. Such features of non-natural bases can be takenadvantage of, and are within the scope of the present invention.

For example, non-natural bases can be used to generate duplexed nucleicacid sequences having a single strand overhang. This can be accomplishedby performing a PCR reaction to detect a target nucleic acid in asample, the target nucleic acid having a first portion and a secondportion, where the reaction system includes all four naturally occurringdNTP's, a first primer that is complementary to the first portion of thetarget nucleic acid, a second primer having a first region and a secondregion, the first region being complementary to the first portion of thetarget nucleic acid, and the second region being noncomplementary to thetarget nucleic acid. The second region of the second primer comprises anon-natural base. The first primer and the first region of the secondprimer hybridize to the target nucleic acid, if present. Several roundsof PCR will produce an amplification product containing (i) adouble-stranded region and (ii) a single-stranded region. Thedouble-stranded region is formed through extension of the first andsecond primers during PCR. The single-stranded region includes the oneor more non-natural bases. The single-stranded region of theamplification product results because the polymerase is not able to forman extension product by polymerization beyond the non-natural base inthe absence of the nucleotide triphosphate of the complementarynon-natural base. In this way, the non-natural base functions tomaintain a single-stranded region of the amplification product.

As mentioned above, the polymerase can, in some instances,misincorporate a base opposite a non-natural base. In this embodiment,the misincorporation takes place because the reaction mix does notinclude a complementary non-natural base. Therefore, if given sufficientamount of time, the polymerase can, in some cases, misincorporate a basethat is present in the reaction mixture opposite the non-natural base.

Amplifying

During PCR, the polymerase enzyme, first primer and second primer areused to generate an amplification product as described herein. One PCRtechnique that can be used is a modified PCR, or Fast-Shot™amplification. As used herein, the term “Fast-Shot™ amplification”refers to a modified polymerase chain reaction.

Traditional PCR methods include the following steps: denaturation, ormelting of double-stranded nucleic acids; annealing of primers; andextension of the primers using a polymerase. This cycle is repeated bydenaturing the extended primers and starting again. The number of copiesof the target sequence in principle grows exponentially. In practice, ittypically doubles with each cycle until reaching a plateau at which moreprimer-template accumulates than the enzyme can extend during the cycle;then the increase in target nucleic acid becomes linear.

Fast-shot amplification is a modified polymerase chain reaction whereinthe extension step, as well as the annealing and melting steps, are veryshort or eliminated. As used herein, when referring to “steps” of PCR, astep is a period of time during which the reaction is maintained at adesired temperature without substantial fluctuation of that temperature.For example, the extension step for a typical PCR is about 30 seconds toabout 60 seconds. The extension step for a Fast-Shot™ amplificationtypically ranges from about 0 seconds to about 20 seconds. Preferably,the extension step is about 1 second or less. In a preferred embodiment,the extension step is eliminated. The time for annealing and meltingsteps for a typical PCR can range from 30 seconds to 60 seconds. Thetime for annealing and melting steps for a Fast-Shot™ amplificationgenerally can range from about 0 seconds to about 60 seconds. ForFast-Shot™ amplification, the annealing and melting steps are typicallyno more than about 2 seconds, preferably about 1 second or less. Whenthe extension step is eliminated, the temperature is cycled between theannealing and melting steps without including an intermediate extensionstep between the annealing and melting temperatures.

Additionally, the limit of how quickly the temperature can be changedfrom the annealing temperature to the melting temperature depends uponthe efficiency of the polymerase in incorporating bases onto anextending primer and the number of bases it must incorporate, which isdetermined by the gap between the primers and the length of the primers.Examples of Fast-Shot™ amplification are shown in the Examples.

The number of Fast-Shot™ amplification cycles required to determine thepresence of a nucleic acid sequence in a sample can vary depending onthe number of target molecules in the sample. In one of the examplesdescribed below, a total of 37 cycles was adequate to detect as littleas 100 target nucleic acid molecules.

Amplification Product

As illustrated, for example, in FIGS. 1, 2, and 9-12, PCR is used togenerate an amplification product 120 comprising a double-strandedregion 122 and a single-stranded region 124. As shown in these figures,the double-stranded region 122 results from extension of the first andsecond primers 106 and 108. As discussed above, the single-strandedregion 124 results from incorporation of a non-natural base in thesecond primer of the invention. The second region 112 of the secondprimer 108 is not complementary to the target nucleic acid 100. Becausethe non-natural base follows base-pairing rules of Watson and Crick andforms bonds with other non-natural bases, as discussed above, thepresence of the non-natural base maintains the second region 112 as asingle-stranded region 124 in the amplification product 120.

In an alternative embodiment, the single-stranded region 124 comprisesmore than one non-natural base. The number of non-natural bases includedin the second region 112 of the second primer 108 can be selected asdesired.

Reporter

As used herein, the term “reporter” refers to a moiety (e.g., anoligonucleotide) that is complementary, and therefore forms a duplexstructure with, the second portion of the second primer. Referring tothe embodiments shown in, for example, FIGS. 1, 2, and 9-12, thereporter comprises a label 128, 132 (154 in FIG. 2) and at least onenon-natural base 130 (152 in FIG. 2) that is complementary to thenon-natural base 114 of the single-stranded region 124. The reporter,preferably, is not complementary to either the first primer or thesecond primer for the polymerase chain reaction. Preferably, the 3′terminus of the reporter is “blocked” to inhibit incorporation of thereporter into a primer extension product. “Blocking” can be achieved byusing non-complementary bases or by adding a chemical moiety such asbiotin or a phosphate group to the 3′ hydroxyl of the last nucleotide,which can, depending upon the selected moiety, serve a dual purpose byalso acting as a label.

Reporters useful in the invention can contain more than one non-naturalbase. The number of non-natural bases included in the reporter can bedetermined by the user and will depend upon such factors as, forexample, the length and base composition of the second region of thesecond primer and the desired hybridization conditions and hybridizationspecificity.

The nucleotide content of the reporter is typically determined by thenucleotide content of the second region of the second primer. That is,the sequence of the reporter is determined by determining the sequenceof the second region of the second primer, and determining thecomplement to that second region, using standard rules developed byWatson and Crick. In one embodiment, for example, the second region ofthe second primer comprises a single non-natural base. In thisembodiment, the reporter would preferably include a single non-naturalbase that is complementary to the non-natural base included in thesecond primer. Likewise, when more than one non-natural base is includedin the second region of the second primer, the sequence of the secondregion determines the complement to that sequence, and the reporter issynthesized accordingly.

Reporters having the same sequence that is capable of hybridizing to thesecond portion of a second primer can be used in a variety of assays,provided that the second portion of the second primer is also the samein those assays. In other words, a “universal” reporter and secondportion of a second primer can be used. The “universal” second portionof the second primer can then be attached to or synthesized as part of asecond primer, where the first portion is specific to the target nucleicacid. This can be used, for example, in kits that are customized by theuser for a desired target nucleic acid.

In other embodiments, within a given assay, it is beneficial to useseveral second primers, each with a different sequence in their secondregions, and several reporters, each having a sequence complementary tothe second portion of one of the several different second primers. Insuch assays it can be beneficial for each reporter to have a differentlabel. In some embodiments, the reporters may be attached by their 3′ends to a discrete region of a solid or other unique support.

The ability of reporters to form sufficiently stable hybrids tooligonucleotides having complementary sequences depends on severalfactors, as discussed above for primers.

In an alternative embodiment, the second primer and the reporter are asingle compound. This embodiment is illustrated in FIG. 4. As shown inFIG. 4A, the target nucleic acid 100 is contacted with a first primer106 and a second primer 108. In this embodiment, the second primercomprises: a first region 110, a second region 112, a linker 180, areporter 190, and a quencher 196. In this embodiment, the linker 180connects the second primer 108 with the reporter 190. The reporter 190comprises a dye 192, a non-natural base 194, and a quencher 196. Thenon-natural base 194 is complementary to the non-natural base 114 of thesecond primer 108. As illustrated in FIG. 4B, the first region 110anneals to the first portion 102 of the target nucleic acid 100. Thelinker 180 comprises a chemical linker that couples the 5′ end of onenucleotide to the 3′ end of another nucleotide. The linker 180 allowsthe reporter 126 to fold back and form base pairs with the second region112 of the second primer 102, as illustrated in FIG. 4B. In oneembodiment, the linker 180 comprises a sequence of nucleotides ofsufficient length to allow the reporter 126 to hybridize with the secondregion 112. Preferably, the nucleotides that comprise the linker 180 inthis embodiment are capable of forming a hairpin loop 182. In anotherembodiment, the temperature at which the reporter 126 hybridizes to thesecond region 112 is lower than the temperature at which the firstregion 110 hybridizes to the second portion 104 of the target 100.

FIG. 4C shows the amplification product 200 that results from extensionof primers 104 and 106 during PCR, or Fast-Shot™ amplification. Theamplification product 200 includes a double-stranded region 202 and asingle-stranded region 204. The reporter 190 anneals to thesingle-stranded region 204 of the amplification product.

As shown in FIG. 4D, the reporter 190 is cleaved by an enzyme, forexample, the polymerase or other suitable enzyme, thus releasing areporter fragment 198. The released reporter fragment 198 includes thedye 192. Release of the dye 192 from proximity of the quencher 196 canbe visualized as described herein. In some embodiments, the reporter 126is hybridized to the second region 112 while the first and secondprimers 106, 108 extend, as illustrated in FIG. 4. This allows thepolymerase to cleave the reporter fragment 198 when the first primer 106has sufficiently extended and can permit the “real time” monitoring ofthe assay during the PCR process without subsequent addition of areporter. The hybridization of the reporter to the second region duringextension of the first and second primers is not, however, a necessaryfeature. Hybridization of the reporter to the second region can occurafter extension in a manner similar to that described for the assayillustrated in FIG. 1.

Label

In accordance with the invention, the reporter comprises a label.Nucleotides and oligonucleotides can be labeled by incorporatingmoieties detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical assays. The method of linking or conjugatingthe label to the nucleotide or oligonucleotide depends on the type oflabel(s) used and the position of the label on the nucleotide oroligonucleotide.

A variety of labels which are appropriate for use in the invention, aswell as methods for their inclusion in the probe, are known in the artand include, but are not limited to, enzyme substrates, radioactiveatoms, fluorescent dyes, chromophores, chemiluminescent labels,electrochemiluminescent labels, such as ORI-TAG™ (Igen), ligands havingspecific binding partners, or any other labels that can interact witheach other to enhance, alter, or diminish a signal. It is understoodthat, should the PCR be practiced using a thermocycler instrument, alabel should be selected to survive the temperature cycling required inthis automated process.

One radioactive atom suitable for a label according to the methods ofthe invention is ³²P. Methods for introducing ³²P into nucleic acids areknown in the art, and include, for example, 5′ labeling with a kinase,or random insertion by nick translation.

It should be understood that the above description is not meant tocategorize the various labels into distinct classes, as the same labelcan serve in several different modes. For example, ¹²⁵I can serve as aradioactive label or as an electron-dense reagent. Further, one cancombine various labels for desired effects. For example, one could labela nucleotide with biotin, and detect its presence with avidin labeledwith ¹²⁵I. Other permutations and possibilities will be apparent tothose of ordinary skill in the art, and are considered within the scopeof the instant invention.

In some situations, it is desirable to use two interactive labels on asingle oligonucleotide with due consideration given for maintaining anappropriate spacing of the labels on the oligonucleotide to permit theseparation of the labels during oligonucleotide hydrolysis. It can besimilarly desirable to use two interactive labels on differentoligonucleotides, such as, for example, the reporter and the secondregion of the second primer. In this embodiment, the reporter and thesecond region are designed to hybridize to each other. Again,consideration is given to maintaining an appropriate spacing of thelabels between the oligonucleotides when hybridized.

One type of interactive label pair is a quencher-dye pair. Preferably,the quencher-dye pair is comprised of a fluorophore and a quencher.Suitable fluorophores include, for example, fluorescein, cascade blue,hexachloro-fluorescein, tetrachloro-fluorescein, TAMRA, ROX, Cy3, Cy3.5,Cy5, Cy5.5,4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid,4,4-difluoro-5,p-methoxyphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid, 4,4-difluoro-5-styryl-4-bora-3a,4-adiaza-5-indacene-propionicacid, 6-carboxy-X-rhodamine, N,N,N′,N′-tetramethyl-6-carboxyrhodamine,Texas Red, Eosin, fluorescein,4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid, 4,4-difluoro-5,p-ethoxyphenyl-4-bora-3a,4a-diaza-s-indacene3-propionic acid and4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-5-indacene-propionic acid.Suitable quenchers include, for example, Dabcyl, QSY7™ (MolecularProbes, Eugene, Oreg.) and the like. In addition, dyes can also be usedas a quencher if they absorb the emitted light of another dye.

The labels can be attached to the nucleotides, including non-naturalbases, or oligonucleotides directly or indirectly by a variety oftechniques. Depending upon the precise type of label used, the label canbe located at the 5′ or 3′ end of the reporter, located internally inthe reporter's nucleotide sequence, or attached to spacer arms extendingfrom the reporter and having various sizes and compositions tofacilitate signal interactions. Using commercially availablephosphoramidite reagents, one can produce oligonucleotides containingfunctional groups (e.g., thiols or primary amines) at either terminus,for example by the coupling of a phosphoramidite dye to the 5′ hydroxylof the 5′ base by the formation of a phosphate bond, or internally, viaan appropriately protected phosphoramidite, and can label them usingprotocols described in, for example, PCR Protocols: A Guide to Methodsand Applications, ed. by Innis et al., Academic Press, Inc., 1990,incorporated herein by reference.

Methods for incorporating oligonucleotide functionalizing reagentshaving one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide reporter sequence, typically at the 5′ terminus, aredescribed in U.S. Pat. No. 4,914,210, incorporated herein by reference.For example, 5′ phosphate group can be incorporated as a radioisotope byusing polynucleotide kinase and [γ³²P]ATP to provide a reporter group.Biotin can be added to the 5′ end by reacting an aminothymidine residue,introduced during synthesis, with an N-hydroxysuccinimide ester ofbiotin.

Labels at the 3′ terminus, for example, can employ polynucleotideterminal transferase to add the desired moiety, such as for example,cordycepin, ³⁵S-dATP, and biotinylated dUTP.

Oligonucleotide derivatives are also available as labels. For example,etheno-dA and etheno-A are known fluorescent adenine nucleotides whichcan be incorporated into a reporter. Similarly, etheno-dC is anotheranalog that can be used in reporter synthesis. The reporters containingsuch nucleotide derivatives can be hydrolyzed to release much morestrongly fluorescent mononucleotides by the polymerase's 5′ to 3′nuclease activity as nucleic acid polymerase extends a primer duringPCR.

In some embodiments, the labeled reporter comprises first and secondlabels wherein the first label is separated from the second label by anuclease-susceptible cleavage site.

The label of the reporter can be positioned at any suitable location ofthe reporter. For example, when the reporter comprises more than onenucleotide, the label can be attached to any suitable nucleotide of thereporter sequence. The label can be positioned at the 5′ terminus of thereporter and separated from the reporter sequence that is complementaryto the target nucleic acid by a non-complementary sequence. In thisembodiment, the reporter comprises a non-natural base that iscomplementary to the non-natural base of the amplification product, anda sequence that is noncomplementary to the second region of the secondprimer, and the label is positioned in the sequence that isnoncomplementary to the second region. Further, the label can beindirectly attached to a nucleotide of the reporter, using a suitablespacer or chemical linker.

In another embodiment, the labeled reporter comprises a pair ofinteractive signal-generating labels effectively positioned on thereporter or on the reporter and a second component of the assay (such asthe second oligonucleotide) so as to quench the generation of detectablesignal when the interactive signal-generating labels are in sufficientlyclose proximity to each other. Preferably, the labels are separated by asite within the reporter that is susceptible to nuclease cleavage,thereby allowing the 5′ to 3′ nuclease activity of the nucleic acidpolymerase to separate the first interactive signal-generating labelfrom the second interactive signal-generating label by cleaving thereporter at the nuclease susceptible site. Separation of the interactivesignal-generating moieties (e.g., cleavage of the reporter to release areporter fragment containing one of the labels) results in theproduction of a detectable signal. Examples of such labels includedye/quencher pairs or two dye pairs (where the emission of one dyestimulates emission by the second dye).

In an exemplified embodiment, the interactive signal generating paircomprises a fluorophore, for example fluorescein,5-[(2-aminoethyl)amino]napthalene-1-sulfonic acid (EDANS),tetramethylrhodamine, or the like, and a quencher that can quench thefluorescent emission of the fluorophore, for example,dimethylaminoazobenzen aminoexal-3-acryinido (Dabcyl). The ordinarilyskilled artisan can select a suitable quencher moiety that will quenchthe emission of the particular fluorophore. In the exemplifiedembodiment, the Dabcyl quencher absorbs the emission of fluorescencefrom the fluorophore moiety. Fluorophore-quencher pairs have beendescribed in Morrison, Detection of Energy Transfer and FluorescenceQuenching in Nonisotopic Probing, Blotting and Sequencing AcademicPress, 1995, incorporated herein by reference.

Alternatively, these interactive signal-generating labels can be used ina detection method where the second region of the second primercomprises at least one non-natural base and a label. The second label ofthe pair is provided by the reporter, which comprises at least onenon-natural base that is complementary to the non-natural base of thesecond primer, and a second label. This embodiment is illustrated inFIG. 6. For example, if a dye/quencher pair is used, hybridization ofthe reporter to or incorporation of the amplification product willresult in a reduction of fluorescence.

Alternatively, the proximity of the two labels can be detected usingfluorescence resonance energy transfer (FRET) or fluorescencepolarization. FRET is a distance-dependent interaction between theelectronic excited states of two dye molecules in which excitation istransferred from a donor molecule to an acceptor molecule withoutemission of a photon. Examples of donor/acceptor dye pairs for FRET areFluorescein/Tetramethylrhodamine, IAEDANS™/Fluorescein (MolecularProbes, Eugene, Oreg.), EDANS™/Dabcyl, Fluorescein/Fluorescein(Molecular Probes, Eugene, Oreg.), BODIPY™ FL/BODIPY™ FL (MolecularProbes, Eugene, Oreg.), and Fluorescein/QSY7™.

Annealing

The reporter is added to the sample at an appropriate time during thedetection method. In the embodiment illustrated in FIG. 1, after PCR hasproduced sufficient amplification product 120, the reporter 126 isannealed to the single stranded region 124 of the amplification product120. In this illustrated embodiment, the reporter 126 comprises a dye128, a quencher 132, and a non-natural base 130 that is complementary tothe non-natural base 114 of the second primer 108. The reporter 126anneals to the sequence corresponding to the second region 112 of thesecond primer 108. The reporter 126 can be added to the reaction mixafter PCR has produced sufficient amplification product 120, or thereporter 126 can be added to the reaction mix prior to PCRamplification. Preferably, the reporter 126 is added to the reaction mixprior to PCR amplification. After amplification, the temperature ispreferably lowered to a temperature lower than the melting temperatureof the reporter/amplification product to allow annealing of the reporterto the single-stranded region of the amplification product. In oneembodiment, the reaction temperature is lowered to about 49° C. or lessduring the step of annealing the reporter to the single-strandedoverhang region Annealing is performed similarly for other embodimentsof the invention including those using other reporters and other typesof labels, as described above. In another embodiment the reporter 126 isannealed at or above the melting temperature of the first and secondprimers 106, 108 and the amplification product 120. This embodiment isparticularly useful when performing “real time” detection of the PCRamplification product

Cleaving

In one embodiment of the invention, after the reporter anneals to theamplification product, a cleavage event occurs to release at least onereporter fragment. The release of the reporter fragment is correlatedwith the presence of the target nucleic acid, as described below. Oncethe reporter anneals to the single-stranded region of the amplificationproduct, this forms a reporter/amplification product complex that isrecognizable by an enzyme that cleaves the complex to release thereporter fragment. The enzymes contemplated for use in this embodimentare generally capable of recognizing a variety of reporter/amplificationproduct complex structures. For example, the 5′ end portion of thereporter 126 can overlap with a sequence of the amplification product,forming a single-stranded overhang region 160 (see FIG. 1D).

In another embodiment, the reporter 126 does not contain an overlappingregion to form a single-stranded overhang region, but rather thereporter forms a nick-like structure when it is annealed to theamplification product (see FIG. 5). In this embodiment, a nick-likestructure is formed in the amplification product, as shown in FIG. 5D.Generally, a “nick” in duplex DNA is the absence of a phosphodiesterbond between two adjacent nucleotides on one strand. As used herein, a“nick-like” structure is formed when there is an absence of thephosphodiester bond between the 5′ terminal nucleotide of the reporter126 and the 3′ terminal nucleotide of the strand 120 c of theamplification product. There are several enzymes, such as, for example,E. coli DNA polymerase I, that are capable of using a nick in duplex DNAas the starting point from which one strand of duplex DNA can bedegraded and replaced by resynthesis of new material.

In this embodiment, the reporter 210 includes non-natural base 216 thatis complementary to the non-natural base 114 of the amplificationproduct, a dye 212, and a quencher 214. The reporter 210 anneals to asingle-stranded portion of the amplification product. Therefore, a nickis produced between the non-natural base 216 and the adjacent nucleotideof the amplification product. In this embodiment, the polymeraserecognizes the nick-like structure formed in the reporter/amplificationproduct complex and cleaves the reporter at that nick site. Cleavage ofthe complex releases the reporter fragment 134, and signal is detected.

While some of the particular structures formed by thereporter/amplification product complex will be discussed in some detail,other reporter/amplification product complexes can be formed to achievecleavage as described herein.

Referring to the embodiment illustrated in FIG. 1D, after annealing, aportion of the 5′ end 160 of the reporter 126 is not annealed to thetarget and is single-stranded. It is understood that any length, inbases, of the single-stranded overhang region 160 is contemplated,provided that the ability of the 5′ to 3′ nuclease activity of thepolymerase to cleave annealed reporter fragments from the amplificationproduct is maintained. For detection in the embodiment exemplified inFIG. 1D, the reaction is continued under conditions sufficient to allowthe 5′ to 3′ nuclease activity of the polymerase to cleave the annealedreporter 126. Cleavage of the reporter 126 produces cleavage fragments134 (containing the label or a part of the label) which can then bedetected (or, alternatively, the remaining reporter/amplificationproduct complex can be detected) and which are indicative of thepresence of the target nucleic acid in the sample. In at least someembodiments, the reporter fragments can include a mixture of mono-, di-,and larger nucleotide fragments.

The nuclease activity of the polymerase cleaves the single-strandedregion 160, releasing a reporter fragment 134 as shown in FIG. 1E. Inthis embodiment, the reporter fragment comprises the dye 128. Release ofthe dye 128 from the amplification product that includes a quencher 132allows detection of the dye. Therefore, release of the reporter fragmentallows detection of the dye as it is released from proximity to thequencher. This, in turn, allows for correlation of the release of thereporter fragment with presence of the target nucleic acid. If instead,the placement of the dye and quencher are reversed and the quencher isreleased with the reporter fragment, the dye on thereporter/amplification product is then detected.

Incorporating

Referring now to FIG. 2, an alternative embodiment of the invention isshown. In this embodiment, the second region 124 of the second primercomprises a non-natural base 124. A non-natural base 152 that iscomplementary to the non-natural base 124 is incorporated into theamplification product using a suitable enzyme. In this embodiment, theincorporation of the reporter is correlated with the presence of thetarget nucleic acid in the sample.

As shown in FIG. 2, the methods of the present invention employ areporter 150; a nucleic acid polymerase (not shown); a first primer 106and a second primer 108. The PCR reaction mixture also contains the fournaturally occurring nucleotide triphosphates (i.e., dATP, dCTP, dGTP,and dTTP) as well as one or more non-natural nucleotide triphosphate (oran oligonucleotide containing a non-natural nucleotide triphosphate) asthe reporter 150. In the illustrated embodiment, the one or morenon-natural nucleotide triphosphates 152 in the reaction mixturecomprises a label 154. The PCR can be a Fast-Shot™ amplification.

The first primer 106 comprises a sequence complementary to a portion ofa target nucleic acid 100 and can hybridize to that portion of thetarget nucleic acid 100. The second primer 108 has a first region 110and a second region 112. The first region 110 comprises a sequencecomplementary to a portion of the target sequence 100. The second region112 of the second primer 108 comprises a non-natural base 114, and thissecond region 112 is not complementary to the target nucleic acid 100.Although only a single nucleotide is illustrated in the second region112, it will be understood that the second region can include additionalnucleotides. Preferably, the non-natural base 114 is located at thejunction between the first region 110 and the second region 112 of thesecond primer 108. In some embodiments, the non-natural base 114 presentin the second region 112 of the second oligonucleotide primer is aniso-C or an iso-G.

In addition to the first primer 106 and second primer 108, the sample iscontacted with a polymerase (not shown), and a polymerase chain reactionis run. If the target nucleic acid 100 is present in the sample, thecomplementary portion of the first primer 106 and the complementaryportion 110 of the second primer 108 anneal to the corresponding regions102, 104 of the target nucleic acid 100 following standard base-pairingrules. Similar to the embodiment shown in FIG. 1, when the primers areannealed to the target, the 3′ terminal nucleotide of the first primer106 is separated from the 3′ terminal nucleotide of the second primer108 by a sequence of nucleotides, or a “gap.” In a preferred embodiment,the first and second primers are designed such that gap of between aboutzero (0) to about five (5) bases on the template nucleic acid existsbetween the 3′ ends of the PCR primers when annealed to the templatenucleic acid.

As shown in FIGS. 2B and 2C, the polymerase is used to synthesize asingle strand 120 a, 120 b from the 3′-OH end of each primer, usingpolymerase chain reaction, or a modified Fast-Shot™ amplification. Thepolymerase chain reaction is allowed to proceed for the desired numberof cycles, to obtain an amplification product 120 shown in FIG. 2C.

As shown in FIG. 2C, the amplification product 120 includes adouble-stranded region 122 and a single-stranded region 124. As shown,the single-stranded region 124 comprises the non-natural base 114 of thesecond primer 108. Although the single-stranded region 124 is shownincluding a single non-natural base, this region can include more thanone non-natural base.

Referring now to FIG. 2D, the amplification product 120 is thencontacted with a reporter 150. The reporter 150 comprises a label 154and a non-natural base 152. The reporter 150 is incorporated into theamplification product opposite the non-natural base 114, as illustratedin FIG. 2E. In one embodiment, the non-natural base 152 of the reporter150 comprises a nucleotide triphosphate base that is complementary tothe non-natural base 114 of the single-stranded region 124 of theamplification product 120. In this embodiment, the PCR reaction includesthe presence of labeled non-natural nucleotide triphosphate base, inaddition to the four naturally occurring nucleotide triphosphate bases(i.e., dATP, dCTP, dGTP, and dTTP). The concentration of non-naturalnucleotide triphosphate base in the PCR reaction can range, for example,from 1 μM to 100 μM.

Suitable enzymes for incorporation of the reporter 150 into theamplification product 120 include, for example, polymerases and ligases.A number of polymerases that are capable of incorporating naturalnucleotides into an extending primer chain can also incorporate anon-natural base into an amplification product opposite a complementarynon-natural base. Typically, class A DNA polymerases; such as Klenow,Tfl, Tth, Tag, Hot Tub, and Bst, are better able than class Bpolymerases; such as Pfu, Tli, Vent exo-, T4, and Pwo, to incorporate anon-natural base. Reverse transcriptases, such as HIV-1, can also beused to incorporate non-natural bases into an extending primer oppositeits complementary non-natural base within a template. In this embodimentthe polymerase can be nuclease deficient or can have reduced nucleaseactivity. While not intended to limit the invention, nuclease deficientpolymerases are expected to be more robust because nuclease activitieshave been shown to interfere with some PCR reactions (Gene, 112(1):29-35(1992) and Science, 260(5109):778-83 (1993).

Presence of the target nucleic acid in the sample is determined bycorrelating the presence of the reporter in the amplification product.Suitable detection and visualization methods are used to detect thetarget nucleic acid. In the illustrated case, for example, presence ofthe target nucleic acid is determined by detecting the label 154, forexample, by fluorescence or other visualization method. Fluorescencepolarization, for example, can be used to detect the incorporation ofthe reporter into the amplification product.

Preferably, in this embodiment, a washing step or a separation step isperformed after incorporation of the reporter 150 into the amplificationproduct 120, and prior to detection. This washing or separation stepwill remove unbound reporter 150 from the system, so that detection ofsignal is dependent upon incorporated reporter. One of skill in the artwould readily appreciate that any known washing or separation steps canbe used in connection with the invention, including size separation bygel electrophoresis, and the like. Alternatively, a washing step is notneeded when fluorescence polarization is used as the method ofdetection.

The reporter 150 used in this embodiment comprises at least onenon-natural base 152. The non-natural base(s) of the reporter preferablyinclude a label 154. The non-natural base(s) 152 of the reporter 150 iscapable of being inserted by the polymerase into the amplificationproduct opposite to the at least one non-natural base 114 of the secondprimer 108 during the PCR amplification.

In another embodiment, illustrated in FIG. 6, the reporter 170 comprisesa non-natural base 172 that is complementary to non-natural base 114 ofthe second primer 108, and a quencher 129. In this embodiment, thenon-natural base 114 of the second primer 108 includes a dye 162. Inthis embodiment, incorporation of the reporter 170 brings the quencher129 into proximity with the dye 162. This, in turn, reduces the signaloutput of the dye 162, and this reduction in signal can be detected andcorrelated with the presence of the target nucleic acid. Suitabledye-quencher pairs are discussed above. Alternatively, a dye-dye paircan be used. When the target nucleic acid is present, PCR creates aduplexed product that places the two dyes in close proximity, and thefluorescent output of the label changes. The change is detectable bybench-top fluorescent plate readers.

The polymerase used in this embodiment can have nuclease activity, canhave reduced nuclease activity, or can be nuclease deficient.Preferably, the polymerase is a thermostable polymerase.

Detection

Detection and analysis of the reporter oligonucleotide fragments can beaccomplished using any methods known in the art. Numerous methods areavailable for the detection of nucleic acids containing any of theabove-listed labels. For example, biotin-labeled oligonucleotide(s) canbe detected using non-isotopic detection methods which employ avidinconjugates such as streptavidin-alkaline phosphatase conjugates.Fluorescein-labeled oligonucleotide(s) can be detected using afluorescein-imager.

In one embodiment the reporter oligonucleotides can be detected withinthe PCR reaction mixture without any further processing. For example,the signal from cleaved oligonucleotides can be resolved from that ofuncleaved oligonucleotides without physical separation. This can beaccomplished, for example by fluorescence polarization analysis where achange in size and therefore rate of rotation in solution of fluorescentmolecules can be detected.

In one embodiment, when the target is present, a duplexed product iscreated that places the first and second labels (e.g., dye/dye pair)into close proximity. When the two labels are in close proximity, thefluorescent output of the reporter molecule label changes. The change isdetectable by most bench-top fluorescent plate readers. Alternatively,the label pair comprises a quencher-label pair in close proximity. Inthis embodiment, the fluorescent output of the reporter molecule labelchanges, and this change is detectable. Other suitable detection methodsare contemplated in this invention.

In another embodiment, the reporter is detected after furtherprocessing. It is contemplated that the reporter oligonucleotidefragments can be separated from the reaction using any of the manytechniques known in the art useful for separating oligonucleotides. Forexample, the reporter oligonucleotide fragments can be separated fromthe reaction mixture by solid phase extraction. The reporteroligonucleotide fragments can be separated by electrophoresis or bymethods other than electrophoresis. For example, biotin-labeledoligonucleotides can be separated from nucleic acid present in thereaction mixture using paramagnetic or magnetic beads, or particleswhich are coated with avidin (or streptavidin). In this manner, thebiotinylated oligonucleotide/avidin-magnetic bead complex can bephysically separated from the other components in the mixture byexposing the complexes to a magnetic field. In one embodiment, reporteroligonucleotide fragments are analyzed by mass spectrometry.

In some embodiments, when amplification is performed and detected on aninstrument capable of reading fluorescence during thermal cycling, theintended PCR product from non-specific PCR products can bedifferentiated. Amplification products other than the intended productscan be formed when there is a limited amount of template nucleic acid.This can be due to a primer dimer formation where the second primer 108is incorporated into a primer dimer with itself or the first primer 106.During primer dimer formation the 3′ ends of the two primers hybridizeand are extended by the nucleic acid polymerase to the 5′ end of eachprimer involved. This creates a substrate that when formed is a perfectsubstrate for the primers involved to exponentially create more of thisnon-specific products in subsequent rounds of amplification. Thereforethe initial formation of the primer dimer does not need to be afavorable interaction since even if it is a very rare event theamplification process can allow the dimer product to overwhelm thereaction, particularly when template nucleic acid is limited or absent.When the second oligonucleotide primer 106 is incorporated into thisproduct a labeled nonstandard base 170 is placed orthogonal to thenonstandard base 114 of the second primer 106. This results in aninteraction between the labels 129 of the reporter and 162 of the secondprimer which would give the same fluorescent output change as in theformation of the intended product 120 as shown in FIG. 6E. Primer dimerproducts are typically shorter in length than the intended product andtherefore have a lower melting temperature. Since the labels are held inclose proximity across the duplex as shown in 6E an event that wouldseparate the two strands would disrupt the interaction of the labels.Increasing the temperature of the reaction which contains the reactionproducts to above the Tm of the duplexed DNAs of the primer dimer andintended product would melt the DNA duplex of the product and disruptthe interaction of the labels giving a measurable change influorescence. By measuring the change in fluorescence while graduallyincreasing the temperature of the reaction subsequent to amplificationand signal generation it is possible to determine the Tm of the intendedproduct as well as that of the nonspecific product.

Nested PCR

Nested PCR can be performed using the method of the invention. By way ofexample, nested PCR can be performed using a first, second, and thirdprimers (or more). The second primer has a first region complementary tothe target sequence and a second region complementary to the reporteroligonucleotide. The first and third primers can hybridize to the targetat higher temperatures than the second primer. A first amplificationproduct can be produced after several PCR cycles are performed wherecycling between denaturation and annealing temperatures allows annealingof the first and third primer to the target nucleic acid, but not thesecond primer. The PCR annealing temperature can subsequently be reducedto allow the first region of the second primer to hybridize to the firstamplification product. Several cycles of PCR at the reduced annealingtemperature can produce a second amplification product between the firstand second primers. The temperature can be lowered to allowhybridization of the reporter oligonucleotide to the second region ofthe second primer.

Use in Detection of DNA Polymorphisms

The methods of the invention are useful for detecting sequencevariations in nucleic acid sequences. As used herein, “sequencevariation” refers to differences in nucleic acid sequence between twonucleic acids. For example, a wild-type gene and a mutant form of thisgene can vary in sequence by the presence of single base substitutionsor deletions or insertions of one or more nucleotides. These two formsof the gene are said to vary in sequence from one another. One exampleof sequence variation is DNA polymorphisms. In an embodiment illustratedin FIG. 7, detection of a single nucleotide polymorphism (SNP) using PCRand requiring no further sample manipulation other than placing the PCRreaction plate onto a fluorescence plate reader is illustrated.Allele-specific reporters or primers are used which contain anallele-specific label. For example, a two allele system might includeallele-specific reporters or primers with labels having differentcolors. The presence of either color indicating the presence of thatallele in the sample and the presence of the combination of the twocolors indicating that both alleles are present in the sample.

In this embodiment, the primers are designed to detect the singlenucleotide polymorphism as follows. Preferably, one of the primers usedcomprises an allele specific primer. Preferably, one of the primerscomprises a non-natural base. In one embodiment, both of these featuresare provided by a single primer. Alternatively, the allele specificprimer is a separate primer from the primer that comprises a non-naturalbase.

As used herein, “allele specific primer” means a primer that iscompletely complementary to a target nucleic acid in a region suspectedto contain a SNP.

The allele specific PCR primers that can be used to discriminate the SNPalleles are designed to be complementary to each allele such that thepolymorphic base of interest is positioned at the 3′ end of the primer.High levels of allelic discrimination are achieved in part by thelimited ability of the polymerase to extend a primer which has anucleotide mismatch at its 3′ end with that of the target DNA, i.e., thecorresponding allele to which the primer is not specific. Additionally,allelic discrimination can be accomplished by placing the mismatch atother positions in the allele specific primer. Generally, the allelespecific position can be anywhere within the primer provided that thepolymerase cannot efficiently extend the primer if there is a mismatch.Preferably, the primers are chosen so that the allele mismatchsufficiently destabilizes hybridization of the allele-specific primer toa target nucleic acid sequence of a different allele for the selectedPCR conditions. In one embodiment, the allele specific position iswithin about 5 bases from the 3′-end of the primer. For example, theallele specific position can be at the 3′-terminal base of the primer.These alternate positions for the allele specific position in the primercan be used to achieve selective amplification in two primary ways: 1)by lowering the Tm of the primer so that it is not hybridized on thetemplate DNA during thermal cycling for the polymerase to extend, or 2)by creating an unfavorable primer/template structure that the polymerasewill not extend. Enhanced specificity is achieved by using Fast-Shot™amplification cycles where the extension stop time, as well as the stoptimes for annealing and melting, are brief or non-existent. In one suchembodiment, the reactions are rapidly cycled between about 90-100° C.and about 50-65° C. with a maximum of about a one-second hold at eachtemperature, thereby leaving the polymerase little time to extendmismatched primers. In an exemplified embodiment, the reaction is cycledbetween about 95° C. and about 58° C. with about a one second hold ateach temperature. This rapid cycling is made possible by generating theshortest possible PCR product by, in general, leaving a gap of aboutzero (0) to about five (5) bases on the template nucleic acid betweenthe 3′ bases of the PCR primers. Preferably, the primers are designed tohave the shortest sequence possible and a Tm of approximately 55-60° C.In one embodiment involving SNP analysis on genomic DNA samples a totalof about 37 cycles was adequate to detect as little as 30 targetmolecules.

One example of an allele specific assay method is illustrated in FIG. 7.It will be recognized that the other assays discussed herein can be usedor modified for allele-specific assays. Referring to FIG. 7A, a sampleis suspected to contain target nucleic acid 100, the target nucleic acid100 including a first portion 102 and a second portion 104. As shown,target nucleic acid 100 is a double-stranded molecule comprised ofstrands 100 b and 100 c.

Referring to FIG. 7B, the sample is contacted with two or moreallele-specific first primers 106 a, 106 b and a second primer 108 asillustrated. One of the allele-specific first primers 106 a iscomplementary to the first portion 102 of the target nucleic acid 100.The other allele specific primer(s) 106 b are not fully complementary tothe first portion 102 of the target nucleic acid 100. The second primer108 includes a first region 110 and a second region 112, the firstregion 110 comprising a sequence that is complementary to the secondportion 104 of the target nucleic acid 100. The second region 112 of thesecond primer 108 includes a non-natural base 114. The second region 112is not complementary to the target nucleic acid 100.

In addition to the first primers and the second primer, the sample isalso contacted with a polymerase and subjected to polymerase chainreaction (PCR), as herein described. If the target nucleic acid 100 ispresent in the sample, the complementary portion of the allele-specificfirst primer 106 a and the complementary portion of the second primer108 anneal to the corresponding regions 102 and 104 of the targetnucleic acid 100 following standard base-pairing rules.

As shown in FIG. 7B, the polymerase is used to synthesize a singlestrand from the 3′-OH end of each primer 106 a, 108, using PCR, orFast-Shot™ amplification. That is, allele specific first primer 106 a isused to synthesize strand 120 c that is complementary to at least aportion of strand 100 c of the target nucleic acid 100, and the secondprimer 108 is used to synthesize strand 120 b that is complementary toat least a portion of strand 100 b of the target nucleic acid 100.Allele-specific first primer 106 b does not substantially extend becauseit is not fully complementary to the target nucleic acid 100. Thepolymerase chain reaction is allowed to proceed for the desired numberof cycles to obtain an amplification product 120 shown in FIG. 7C. Theassay then proceeds as described for the assay illustrated in FIG. 1.

Kits

Reagents employed in the methods of the invention can be packaged intodiagnostic kits. Diagnostic kits include labeled reporter, first primer,and second primer. In some embodiments the kit includes non-naturalbases capable of being incorporated into an elongating oligonucleotideby a polymerase. In one embodiment, the non-natural bases are labeled.If the oligonucleotide and non-natural base are unlabeled, the specificlabeling reagents can also be included in the kit. The kit can alsocontain other suitably packaged reagents and materials needed foramplification, for example, buffers, dNTPs, or polymerizing enzymes, andfor detection analysis, for example, enzymes and solid phaseextractants.

Reagents useful for the methods of the invention can be stored insolution or can be lyophilized. When lyophilized, some or all of thereagents can be readily stored in microtiter plate wells for easy useafter reconstitution. It is contemplated that any method forlyophilizing reagents known in the art would be suitable for preparingdried down reagents useful for the methods of the invention.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLES Example 1 Primer Design

The symbols indicated in the sequence of the nucleic acid components areas follows: A=deoxyadenylate; T=deoxythymidylate; C=deoxycytidylate;G=deoxyguanylate; X=deoxy-iso-cytosine (d-isoC); Y=deoxy-iso-guanine(d-isoG); P=nucleotide of first primer complementary to polymorphicnucleotide in target nucleic acid; B=3′ modification of reporter nucleicacid by addition of BiotinTEG CPG (Glen Research, Sterling, Va.) to 3′end that functions to block nucleic acid polymerase and extension of thereporter; Q=signal quenching element(5′-5-[(N-4′-carboxy-4(dimethylamino)-azobenzene)-aminohexyl-3-acrylimido]-2′-deoxyUridine(Dabcyl dT; Glen Research, Sterling, Va.) incorporated into reporter byaddition of

5′-Dimethoxytrityloxy-5-[(N-4′-carboxy-4(dimethylamino)-azobenzene)-aminohexyl-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Dabcyl dT; Glen Research, Sterling, Va.); FAM=Signal generating element(6-carboxyfluorescein (6-FAM); Glen Research, Sterling, Va.).Underlining indicates the portion of the nucleic acid component that isnot complimentary to the template.

The designs of the nucleic acid components are shown below:

Nucleic Acid Component Sequence SEQ ID NO Reporter5′-FAM-TYQCCTGTCTGC-3′ SEQ ID NO: 1 First Primer 5′-GGCCAGCATAAGCCM-3′SEQ ID NO: 2 Second Primer 3′-GTTGCTTTTGTCGACTACCAXAGGACAGACG-5′SEQ ID NO: 3

The first primer was designed to have a T_(m) of approximately 60° C.The second primer was designed to have a T_(m) of approximately 61° C.T_(m) can be estimated using a variety of known techniques includingPeyret et al., Biochemistry, 38:3468-77 (1999), incorporated herein byreference.

Hybridization conditions were as follows:

Component Concentration Na⁺ 0.04 mol/L Mg²⁺ 0.002 mol/L First Primer 0.2μmol/L Second Primer 4.0 μmol/L

A 3′ G was avoided in designing the allele specific primer due to thetendency of Taq polymerase to extend G mismatches.

The first region, or the 3′ end, of the second primer was complimentaryto the second portion, or downstream region, of a target nucleic acidsequence. The location of the second primer on the target nucleic acidsequence provided a gap, or a region, of the target nucleic acid that isbetween the 3′ ends of the first and second primers, which can be from 0to about 5 nucleotides. In synthesizing the second primer, incorporationof the iso-cytosine nucleoside, in the second region of the primer wascarried out using standard DNA synthesis conditions.

Example 2 Allele Specific PCR

The following nucleic acid components were used in a fluorescence-basedPCR reaction:

Nucleic Acid Component Sequence SEQ ID NO Reporter5′-FAM-TYQCCTGTCTGC-3′ SEQ ID NO: 1 First Primer, 5′-GGCCAGCATAAGCCC-3′SEQ ID NO: 4 C specific First Primer, 5′-GGCCAGCATAAGCCA-3′ SEQ ID NO: 5A specific Second 3′-GTTGCTTTTGTCGACTACCAXAGGACAGACG-5′ SEQ ID NO: 3Primer Template, G 3′-GGGAATGCAGTTCGATCAGTGAAACGAACGTTC SEQ ID NO: 6TGACCTTTAAGT-5′ 5′-CCCTTACGTCAAGCTAGTCACTTTGCTTGCAAGA SEQ ID NO: 7CTGGAAATTCA-3′ Template, A 3′-GGGAATGCAGTTCGATCAGTTAAACGAACGTTCSEQ ID NO: 8 TGACCTTTAAGT-5′ 5′-CCCTTACGTCAAGCTAGTCAATTTGCTTGCAAGASEQ ID NO: 9 CTGGAAATTCA-3′

The working concentration (1×) of components in PCR reaction forindividual 20 μl PCR reaction volumes is shown below:

Component 1X Conc. Tris pH 8.0 10 mM Bovine Serum Albumin 0.01% Triton ™X-100 0.01% Herring Sperm DNA 0.1 μg/ml Potassium acetate 40 mM MgC1₂ 2mM Amplitaq Gold ™ DNA 1 U/rxn polymerase dATP 50 μM dGTP 50 μM dCTP 50μM dTTP 50 μM First Primer 0.2 μM Second Primer (A or B) 0.2 μM Reporter0.4 μM

All components were thawed on ice and gently mixed together. A 10×PCRBuffer was prepared and composed of 100 mM Tris pH 8.0, 0.1% BSA, 0.1%Triton X-100, 1 mg/ml degraded herring sperm DNA (Sigma D-3159), 400 mMpotassium acetate, and 20 mM MgCl₂. A master mix and an allele specificmix were prepared by adding the reagents in the proportions indicatedbelow:

Master Mix Volume per Concentration Component Reaction (μL) in ReactiondH₂O 11.36 — 10X PCR Buffer 2 1X dNTPs 25 mM 0.04 50 μM Reporter 0.2 0.4μM Second primer 0.2 0.2 μM Amplitaq Gold ™ DNA 0.2 1 U polymerase

The final volume of the reaction was 20 μL. 5 μL of target nucleic acidwas added to 15 μL, of the combined Master Mix and first primer. Thetarget nucleic acid volume can be increased or decreased according toend user needs by adjusting the amount of water added in the Master Mix.5 μL, is a convenient volume to deliver with a multichannel pipetor. TheAllele Specific Mixes were prepared as shown below:

Allele Specific Mixes Volume per Concentration Component Reaction (μL)in Reaction First Primer 1 0.2 μM Master Mix 14 —

The assay plates were prepared as follows: 15 μL of an allele specificmix (as defined above) was aliquoted into a 96-well assay plate. (Anallele specific mix can be prepared and run for each specific firstprimer that is to be used in the assay). The target nucleic acid sampleswere added in duplicate in a volume of 5 μL to each well containing anallele specific mix. A certain number of wells were reserved ascontrols; a negative control (no target nucleic acid) should be run witheach of the allele specific mixes. Subsequent to the target nucleic acidaddition, the reactions were overlaid with 20 μL of mineral oil and theassay plate was transferred to a DNA thermal cycler. Hands on time ofthis procedure was greatly reduced by the use of a multichannel pipetor.

The thermal cycling parameters for the assay plates are shown below:

Cycle # Step Temp Time  1 1 95° C. 12 min. 2-38 1 95° C. 1 sec. 2 58° C.1 sec. 39 1 49° C. 20 min.

Following PCR cycling reactions, the assay plates were tested foremission of a fluorescence signal. The assay plates were transferred toa PerSeptive Biosystems Cytofluor™ 4000 fluorescence plate reader andthe instrument set to read from the top of the plate. The parameters forthe plate reader were as follows: excitation filter settings at 485±10nm; emission filter settings at 530±12.5 nm, and PMT gain set to 50. Thesamples were then read.

Table 1 shows readings obtained using the allele specific primers.

TABLE 1 Copy number, target C-specific first primer A-specific firstprimer nucleic acid 3000 300 30 3000 300 30 RFUs: G-target 1847 1071 1311 1 nucleic acid RFUs: T-target 7 1 1464 1066 176 nucleic acid RFU =Relative Fluorescence Unit

Example 3 Comparison of “Fast-Shot™” Amplification Versus Standard PCRin SNP Detection

This example shows the relative levels of allelic discrimination between“Fast-Shot™” amplification and traditional PCR cycling parameters byvarying the target levels over three orders of magnitude. Fast-Shot™amplification involves cycling between the denaturation and annealingtemperatures of the primers with stops at these temperatures for veryshort periods of time (for example, 1 second).

The following nucleic acid components were used:

Nucleic Acid Component Sequence SEQ ID NO First5′-CCCTTACGTCAAGCTAGTCAC-3′ SEQ ID NO: 10 Primer, C specific First5′-CCCTTACGTCAAGCTAGTCAA-3′ SEQ ID NO: 11 Primer, A specific Second3′-ACGAACGTTCTGACCTTTAAGT-FAM-5′ SEQ ID NO: 12 Primer Template,3′-GGGAATGCAGTTCGATCAGTGAAACGAACGTTCTG SEQ ID NO: 6 G ACCTTTAAGT-5′5′-CCCTTACGTCAAGCTAGTCACTTTGCTTGCAAGACT SEQ ID NO: 7 GGAAATTCA-3′Template, 3′-GGGAATGCAGTTCGATCAGTTAAACGAACGTTCTG SEQ ID NO: 8 AACCTTTAAGT-5′ 5′-CCCTTACGTCAAGCTAGTCAATTTGCTTGCAAGA SEQ ID NO: 9CTGGAAATTCA-3′

The template nucleic acid concentration was in attomol range. Theworking concentration (1×) of components in PCR reaction for individual20 μl PCR reaction volumes are shown below.

Component 1X Conc. Tris pH 8.0 10 mM Bovine Serum Albumin 0.01% Triton ™X-100 0.01% Herring Sperm DNA 0.1 μg/ml Potassium acetate 40 mM MgCl₂ 2mM Amplitaq ™ Gold 1 U/rxn or Amplitaq ™ Stoffel DNA polymerase dATP 50μM dGTP 50 μM dCTP 50 μM dTTP 50 μM First Primer 0.2 μM Second Primer (Aor B) 0.2 μM Reporter 0.4 μM

The PCR reactions were prepared using the same procedure as described inExample 2.

The following PCR parameters were utilized for “fast shot” PCR:

Cycle # Step Temp Time 1-25 1 95° C. 1 sec. 2 61° C. 1 sec.

The following PCR parameters were utilized for traditional PCR:

Cycle # Step Temp Time 1-25 1 95° C. 30 seconds 2 61° C. 30 seconds

Amplitaq™ Gold is a 5′→3′ exonuclease positive Taq polymerase andAmplitaq™ Stoffel is a 5′→3′ exonuclease deficient Taq polymerase.

The data shown in Table 2 shows the relative levels of allelicdiscrimination between “Fast-Shot™” amplification and traditional PCRcycling parameters by varying the target nucleic acid levels over threeorders of magnitude. By comparison of the band intensities of thespecific reactions (C-primer/G-target, and A-Primer/T-target) and themismatched reactions (C-primer/T-target, and A-Primer/G-target), levelsof allelic discrimination can be determined. Table 2 summarizes thelevels of discrimination seen in these experiments.

TABLE 2 A-specific first primer C-specific first primer Fast-shot ™Traditional Fast-shot ™ Traditional amplification PCR amplification PCRAmplitaq ™ >1:1000 >1:1000 1:1000 1:1  Gold Amplitaq ™ >1:1000 >1:10001:1000 1:100 Stoffel

As shown in the results, certain 3′ mismatches are more readily extendedby nucleic acid polymerases. In this case under traditional PCRparameters the C/T mismatch is extended to a much greater extent thanthe A/G mismatch by both 5′→3′ exonuclease containing Amplitaq™ Gold and5′→3′ exonuclease deficient Amplitaq™ Stoffel DNA polymerases. Byemploying “Fast-Shot™” amplification a 1:1000 level of discriminationbetween both alleles is achieved using either enzyme.

Example 4 PCR and Reporter Annealing

The following nucleic acids were used for fluorescence-based PCRreactions:

Nucleic Acid Component Sequence SEQ ID NO Reporter A5′-FAM-TYQCCTGTGTGC-3′ SEQ ID NO: 1 Reporter B 5′-FAM-XYQCCTGTCTGC-3′SEQ ID NO: 13 First 5′-CTCATGGACCCCCATAC-3′ SEQ ID NO: 14 Primer Second3′-GGTGCGAGGTCAATCGAXAGGACAGACG-5′ SEQ ID NO: 15 Primer A Second3′-GGTGCGAGGTCAATCGYXAGGACAGACG-5′ SEQ ID NO: 16 primer B Template5′-CCTCATGGACCCCCATACATATTGTCCACGCT- SEQ ID NO: 17 CCGTTAGC-3′

2 fM of synthetic template controls in 2 μg/ml herring sperm DNA, and 2mM MOPS pH 7.0 were used.

The reaction components for the following reaction are shown below.

Component 1X Conc. Tris pH 8.0 10 mM Bovine Serum Albumin 0.01% Triton ™X-100 0.01% Herring Sperm DNA 0.1 μg/ml Potassium acetate 40 mM MgCl₂ 2mM Amplitaq ™ Gold 1 U/rxn DNA polymerase dATP 50 μM dGTP 50 μM dCTP 50μM dTTP 50 μM First Primer 0.2 μM Second Primer (A or B) 0.2 μM Reporter0.4 μM

AMPLITAQ GOLD™ 5 U/μl was obtained from Perkin Elmer.

Reagents were thawed and gently mixed and two master mixes wereprepared. One master mix (A) contained the second primer A and thereporter A. The other master mix (B), contained the second primer B andthe reporter B. The Master Mixes were prepared as shown below.

Master Mix Volume per Concentration Component Reaction (μL) in ReactiondH₂O 11.36 — 10X PCR Buffer 2 1X dNTPs 25 mM 0.04 50 μM Reporter 0.2 0.4μM Second primer 0.2 0.2 μM Amplitaq Gold ™ DNA 0.2 1 U polymerase Firstprimer 1 0.2 μM

The final volume of the reaction was 20 μl. 5 μl of target nucleic acidwas added to 15 μl of combined Master Mix and First primer. The targetnucleic acid volume can be increased or decreased according to end userneeds by adjusting the amount of water added in the Master Mix.

Assay plates were prepared as follows: 15 μl of a master mix wasaliquoted to wells of a 96-well assay plate (Low Profile Multiplate™ 96well; MJ Research, MLL-9601). Target nucleic acid samples were added induplicate in a volume of 5 μl to wells containing the master mix. Tosome wells, 5 μl of water, rather than target nucleic acid, was added asa negative control. Subsequent to target nucleic acid or negativecontrol addition, the reactions were overlaid with 20 μl of mineral oilMineral Oil (light white oil; Sigma, M-3516) and the assay plate wastransferred to a DNA thermal cycler.

Thermal cycling parameters for the assay plates are shown below:

Cycle # Step Temp Time 1. 1 95° C. 12 min.  2.-38. 1 95° C. 1 sec. 2 58°C. 1 sec. 39a. 1 49° C. 20 min. 39b. 1 51° C. 20 min. 39c. 1 53° C. 20min. 39d. 1 55° C. 20 min. 40.  1 4° C. hold

As an additional control, some of the samples were not subjected to PCRthermal cycling.

Following PCR cycling reactions, the assay plates were tested foremission of fluorescence signal. The assay plates were transferred to aPerSeptive Biosystems Cytofluor™ 4000 fluorescence plate reader and theinstrument set to read from the top of the plate. The parameters for theplate reader are as follows: excitation filter settings at 485±10 nm;emission filter settings at 530±12.5 nm, PMT gain set to 50. The sampleswere then read. The assay plates were also tested for the emission offluorescent signal prior to PCR amplification as a control.

The samples were subsequently run on 10% native polyacrylamide gelelectrophoresis (PAGE) followed by ethidium bromide staining to detectthe presence of an amplification product and to confirm the fluorescencereadings taken from the assay plates.

The results of the fluorescence detection of the target nucleic acidtemplate is shown Table 3. The amplification product was also detectedby ethidium bromide staining after PAGE. In Table 3, a (−) indicates theabsence and a (+) indicates the presence of the template or process ofthermal cycling. The numbers indicated in Table 3 indicate the relativefluorescence units (RFUs).

TABLE 3 Master A A A A A A B B B B B B mix Target + + − − − − + + − − −− Thermal + + + + − − + + + + − − cycling T = 0  198  203 208 206 205208 170 173 172 173 166 170 49° C. 1272 1440 234 243 240 235 470 494 382398 264 267 51° C. 1543 1725 248 252 257 244 603 644 387 403 279 273 53°C. 1540 1666 250 253 255 250 718 773 382 403 283 284 55° C. 1590 1724261 263 273 264 806 866 380 399 285 287

As shown in Table 3, several second primers and reporters can besuccessfully used to detect the presence of a target nucleic acidsequence present in a sample. The second primer/reporter combination inmaster mix A produced a more robust signal than the secondprimer/reporter combination in master mix B. (It will also be recognizedthat master mix A included more PCR product than master mix B, whichaccounts for some of the difference in signal intensity.) The differencein signal intensity appeared to be greater at lower temperatures.However, the second primer and reporter in master mix B produced asignal above background at all hold temperatures, and therefore weresufficient for detection and quantification of the target nucleic acidsequence.

PCR products were separated by PAGE and stained with ethidium bromide orscanned for fluorescein fluorescence using a Molecular Dynamics(Sunnyvale, Calif.) 595 Fluoroimager™. For both the master mix A and themaster mix B reactions, an amplification product was detected byethidium bromide staining after PAGE in lanes corresponding to reactionscontaining template (+). An amplification product was not seen in lanescorresponding to reactions not containing a nucleic acid template (−).These results confirmed that the increased fluorescence signals detectedin the above assay were due to presence of nucleic acid target sequencein the sample.

The differences in the signal intensity between the secondprimer/reporter combination of master mix A and second primer/reportercombination of master mix B were likely due to the degradation of thenon-natural base, isoC, during the reaction. The non-natural base, isoC,tends to degrade at high temperatures in solutions containingnucleophiles, such as solutions containing Tris buffer. However, theresults presented above do show that isoC is suitable for use in Trisbuffer at high temperatures.

To optimize the efficiency of the methods according to the invention,polymerases that do not require a hot start activation and buffers thatare non-nucleophilic in nature should be used when the non-natural base,isoC, is used.

Example 5 Dry Down Plate Preparation

Some or all of the reagents necessary for the methods of the inventioncan be dried down for convenient storage and ease of use. For example,reactions can be set up as master mixes containing 40 mM Potassiumacetate, 20 mM MgCl₂, 50 μM dNTPs (dATP, dCTP, dGTP, dTTP), 1unit/reaction AMPLITAQ GOLD™ polymerase, a sugar as described below, and8 μM reporter. The Master Mix can then be aliquoted into the wells of 96well microtiter plates and dried in a SPEEDVAC™ (Savant Instruments,Holbrook, N.Y.) for 45-50 minutes (no heat). After desiccation, platescan be covered with MICROSEAL A™ film (MJ Research, Waltham, Mass.)placed in a vacuum bag with 1 DESIPAK™ (Trocken, Germany), and the bagcan be filled with argon and sealed with a FOOD SAVER™ (Tilia, SanFrancisco, Calif.). Various sugars (Mannose, Raffinose, Sucrose, andTrehalose (Sigma, St. Louis, Mo.)) at various concentrations (1%, 2%,5%, and 10% by weight) can be used.

Reaction mixes can be reconstituted in water containing nucleic acidtarget, first primer, second primer, and optionally reporter. Thereaction mixes can then be subjected to PCR

In this way, the dried down reagents can be readily reconstituted andsuccessfully used in PCR assays. Such lyophilized reagents can be storedat room temperature for extended periods of time. Some or all of thereagents can be dried down. Some or all of the lyophilized reagentsnecessary for a given method of the present invention can be stored inwells of microtiter plates for later use after reconstitution.

Example 6 Assay Including PCR Incorporation of Non-Natural Base

The following example illustrates a method for monitoring theaccumulation of PCR product by quenching the signal of a label on thesecond primer by site specific incorporation (SSI) of a nucleotidetriphosphate across the DNA duplex at a position near the label of thesecond primer. The labeled nucleotide triphosphate is incorporated intothe elongating first primer during PCR extension. The label on thelabeled nucleotide triphosphate is capable of quenching the label on thesecond primer. Alternatively fluorescence energy transfer (FRET) can beobserved between the label of the second primer (donor dye) and thelabel of the reporter (acceptor dye). Detection of PCR product can beobserved by exciting the donor dye and reading the emission of theincorporated acceptor dye.

The following nucleic acid components were used in the PCR reaction:

Nucleic Acid Component Sequence SEQ ID NO: First Primer5′-GTYATYTGCG-c3-TCGTGCGGTGCGTC-3′ SEQ ID NO: 18 Second3′-TGTGTCGTGTCGTCCGAT-FAM 5′ SEQ ID NO: 19 Primer A Second3′-TGTGTCGTGTCGTCCGXT-FAM 5′ SEQ ID NO: 20 Primer B Template5′-TCGTGCGGTGCGTCACACAGCACAGCAGGC-3′ SEQ ID NO: 21

“c3” indicates a propyl spacer which was chemically installed in placeof a nucleotide during synthesis of the first primer. Thephosphoramidite used in the synthesis of the first primer was3-O-Dimethyltrityl-propyl-1-[2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Spacer Phosphoramidite C3; Glen Research, Sterling, Va.). Optionally,an oligonucleotide containing an identical nucleotide sequence,including nucleotide modifications, but lacking the propyl spacer can beused as a substitute for the first primer in the PCR reaction.

In design of these systems it is preferred that the labeled nucleotidetriphosphate is complementary to a base near the label of the secondprimer A or B. When using a naturally occurring nucleotide base that islabeled, the ability to incorporate such a complementary base near thelabel of the second primer A or B is possible only in a limited numberof cases. This is because all four naturally occurring nucleotide basesare likely to be incorporated at other positions. By using labelednon-natural bases, such as labeled isoG and isoC for example, thelabeled non-natural base will be incorporated only opposite to acomplimentary non-natural base, which can be placed near the label ofthe second primer A or B.

Systems for using labeled non-natural bases and naturally occurringnucleotide triphosphates utilize a naturally occurring nucleotide (dTTP)labeled a quencher dye (QSY7™) in an assay to detect or quantify theamount of target nucleic acid (template) present in a sample. For thisexample the labeled, non-natural nucleotide was incorporated into thefirst primer during PCR extension at a position opposite to, and near,the label (FAM) of the second primer A. A system for using a non-naturalbase, IsoG (dG_(iso)TP), labeled with a quencher dye (QSY7™) in an assayto detect or quantify the amount of target nucleic acid (template)present in a sample was also performed. For this example the labeled,non-natural nucleotide was incorporated into the first primer during PCRextension at a position opposite to isoC(X), which is near the label(FAM) of the second primer B. The chemical structures of the QSY7™ dTTPand QSY7™ dG_(iso)TP are shown below.

A PCR reaction was performed to demonstrate fluorescence quenching bysite specific incorporation in PCR. PCR conditions: 0.2 μM first primer,0.2 μM second primer A, 0.4 μM template nucleic acid, 50 μM dATP, dGTP,and dCTP, 10 mM Tris pH 8, 0.1% BSA, 0.1% Triton™ X-100, 0.1 μg/μldegraded herring sperm DNA, 40 mM KAc, 2 mM MgCl₂, 1 unit Klentaq™ DNApolymerase (Ab Peptides, St/ Louis, Mo.), and 0 or 3.9 uM QSY7™ dTTP ina 25 μl reaction volume.

The PCR conditions used are shown below.

Cycle # Step Temp Time  1. 1 95° C. 2 min. 2.-36. 1 95° C. 1 sec. 2 60°C. 1 sec. 37. 1 70° C. 6 sec.

Reactions were analyzed for fluorescence on a Cytofluor™ 4000fluorescence plate reader (485 nm excitation/530 nm emission) and by gelelectrophoresis. The hold time of 6 seconds at 70° C. was used to obtainan accurate fluorescence reading. The results are presented in Table 4.

PCR reactions were resolved on a 10% native polyacrylamide gel scannedfor 6FAM using a Typhoon™ fluorescence scanner (Molecular Dynamics,Sunnyvale, Calif.). The relative fluorescent units (RFU's) contained inproduct bands were 1,325,644 for (+) QSY7™ dTTP reaction and 41,462,945for (−)QSY7™ dTTP reaction. The polyacrylamide gel was also stained withethidium bromide (50 μg/ml in 10 mM Tris-HCl, 1 mM EDTA). Quantitationof product bands from ethidium bromide staining revealed 21,993 RFU'sfor the (+)QSY7™ dTTP reaction and 25,537 RFU's for the (−)QSY7™ dTTPreaction.

Table 4 shows net RFU's read in PCR reaction wells prior to and after 35cycles of PCR.

TABLE 4 PCR Cycles (+)QSY7 ™ (−)QSY7 ™ 0 3726 3836 35 1200 4490

These results show a 27 fold reduction of fluorescence intensity whenthe labeled nucleotide triphosphate (QSY7™ dTTP) is incorporated into aduplex across from the fluoroscein label (FAM) of the second primerduring PCR.

The nucleic acid components in this example were also utilized todemonstrate “real time” monitoring of PCR product accumulation by sitespecific incorporation (SSI).

The PCR conditions were as follows: 0.2 μM of first primer, 0.2 μM ofsecond primer A, 0.33 μM of template, 50 μM dATP, dGTP, and dCTP, 10 mMTris pH 8, 0.1% BSA, 0.1% Triton X-100, 0.1 μg/μl degraded herring spermDNA, 40 mM KAc, 2 mM MgCl₂, 1 unit Klentaq™ DNA polymerase (Ab Peptides,St/ Louis, Mo.), and 0 or 3 uM QSY7™ dTTP in a 15 μl reaction volume.

PCR Conditions:

Cycle # Step Temp Time 1. 1 95° C. 2 min. 2.-X.* 1 95° C. 1 sec. 2 60°C. 1 sec. X.* + 1. 1 70° C. 6 sec. (*X = 6, 11, 16, 21, 26, 31, or 36)

Reactions were analyzed for fluorescence (485 nm excitation/530 nmemission) on a Cytofluor™ 4000 fluorescence plate reader (PE Biosystems,Foster City, Calif.) and by gel electrophoresis. FIG. 14 illustrates theresults.

FIG. 14 shows the relative fluorescence vs. PCR cycle number. QSY7™ dTTPcontaining reactions were also examined by gel electrophoresis (5 μl ona 10% native polyacrylamide gel). Staining of the gel with ethidiumbromide (50 μg/ml in 10 mM Tris-HCl, 1 mM EDTA) indicated accumulationof the expected product. Fluorescence of the QSY7™ dTTP containingreactions agree with the appearance and accumulation of PCR productsrevealed by gel analysis. These results also showed that correlatingrounds of PCR versus quenching to target concentration can quantify theamount of target present in a sample. For example, the more target thatis present, the faster quenching will occur.

A hold time of 6 seconds at 70° C. was used for an accurate fluorescencemeasurement in the Example described above. Such a hold time is notrequired for the requisite number of bases to be incorporated across thegap between primers and across the primer. If fluorescence is not to bemeasured, no hold time at 70° C. is needed. If a hold time is requiredfor obtaining a fluorescence reading, or any other suitable measurement,it is preferred that the hold temperature be a temperature above whichthe first primer can effectively hybridize to the target sequence. Inone embodiment, the hold temperature is greater than 10° C. over themelting temperature of the first primer.

Example 7 Synthesis of Labeled Non-Natural Bases

Labeled non-natural bases suitable for the methods and kits of theinvention can be made by a variety of methods. Two synthesis schemes areprovided for labeled-deoxyisoGuanosine 5′-Triphosphates: Process A isillustrated in FIG. 15 and the compounds (compounds 1-8, including8_(a)-8_(d)) of Process A are described in Section A; Process B isillustrated in FIG. 16 and the compounds (compounds 9-18) of Process Bare described in Section B.

Section A

For the following chemical reactions involving the synthesis oflabeled—deoxyisoGuanosine 5′-Triphosphates using Process A, Sephadex™DEAE cellulose, omega-aminobutyl agarose and tributylammoniumpyrophosphate were purchased from Sigma; biotin 2-nitrophenyl ester,6-carboxyfluorescein N-hydroxysuccinimide ester, and6-carboxytetramethylrhodamine N-hydroxysuccinimide ester were purchasedfrom Berry & Associates; QSY7™ N-hydroxysuccinimide ester was purchasedfrom Molecular Probes; all other chemicals were purchased from AldrichChemical Co. or Fisher Chemical Co. and were used without furtherpurification. Solvents were dried over 4 Å molecular sieves. Reactionswere carried out under dry argon in an oven-dry glass system.“Evaporation” refers to removal of volatile solvents with a membranepump. Column chromatography was performed with silica gel (230-425mesh).

6-(6-Aminohexyl)-amino-2-chloropurine 2′-deoxy-3′,5′-di-tolulriboside 2

2,6-Dichloro-2′-deoxy-3′,5′-ditoluoylriboside 1 (1 equiv., 5 mmol, 2.705g), dissolved in DMF (100 ml), was added at room temperature to astirred solution of hexamethylenediamine (20 equiv., 100 mmol, 11.60 g)in 200 ml DMF over 40 minutes. The solution was stirred at 50° C. for2.5 hours, then cooled to room temperature, concentrated and the residueextracted (water/ethyl acetate). The organic layer was washed with water(5×50 ml), dried (Na₂SO₄), and the solvent was evaporated to give 2.673g (4.308 mmol, 86%) product 2 as a foam.

6-(6-Aminohexyl)-amino-2-chloropurine 2′-deoxyriboside 3

The above-obtained compound 2 (4.308 mmol, 2.673 g) was dissolved in 20ml methanol, saturated at 0° C. with ammonia, and placed in a sealedtube. It was heated at 80° C. for 1 hour and cooled to 0° C. The tubewas opened and the solvent evaporated under membrane pump vacuum. Theresidue was treated with ether/hexane three times, and the obtainedpowder was dried in vacuum and used in next step without furtherpurification.

6-(6-Aminohexyl)-amino-2-phenoxypurine 2′-deoxyriboside 4

The above-obtained powder (max. 4.308 mmol) was dissolved in DMF (15 ml)and a solution of NaH (12 equiv., 51.69 mmol, 2.068 g of a 60%dispersion in mineral oil) in benzylalcohol (43 ml) was added. It wasstirred at 100° C. for 2 hours and cooled to room temperature. Aceticacid was then added (12 equiv.) to neutralize the reaction mixture. Theresultant solution was filtered over Celite™, the filtrate wasevaporated, and the obtained residue was used in the next step withoutfurther purification.

6-(6-Trifluoroacetylamidohexyl)-amino-2-phenoxypurine 2′-deoxyriboside 5

The above-obtained product was dissolved in a mixture of methanol (30ml)/ethyl trifluoroacetate (30 ml) and stirred at room temperature for24 hours. The solvent and excess ethyl trifluoroacetate was removed byevaporation, and the residue was purified by column chromatography usinga one step gradient of 1.5% methanol in chloroform, then 17.5% methanolin chloroform. Yield: 626 mg (1.134 mmol, 26% for 3 steps).

6-(6-Trifluoroacetylamidohexyl)-amino-3-phenoxypurine 2′-deoxyriboside5′-triphosphate 6

1,2,4-Triazole (4.5 equiv., 0.585 mmol, 40 mg) was dissolved in amixture of 0.5 ml acetonitrile/4.5 equiv. triethylamine (0.585 mmol,0.081 ml), and the flask was placed in an ice bath. Phosphorusoxychloride (1.5 equiv., 0.195 mmol, 0.018 ml) was added, and it wasstirred at room temperature for 30 minutes. It was filtrated, the solidwas washed with a minimum amount of acetonitrile, and the filtrate wasadded to compound 5 (1 equiv., 0.13 mmol, 72 mg). It was stirred for 30minutes at room temperature, then a solution of tributylammoniumpyrophosphate (89 mg) in DMF (2 ml) was added and stirring continued for19 hours. Then, water (1 ml) was added to hydrolyze the remainingtriazolide group. After stirring for 30 minutes, the reaction mixturewas concentrated in vacuum at 30° C. and purified by columnchromatography on DEAE cellulose using a gradient of 0.05M-0.5M TEABbuffer. The product elutes at a buffer concentration of 0.4-0.5M. Thefractions containing the product were evaporated at 30° C. and usedfurther.

N⁶-(6-Aminohexyl)-2′-deoxyisoGuanosine 5′-triphosphate 7

The above-obtained compound was evaporated with methanol and dissolvedin methanol (5 ml). Pd/C (10 weight %, 10 mg) and HCOONH₄ (63 mg) wereadded and it was stirred under reflux for 45 minutes. Then it was cooledto room temperature, filtered from the catalyst, the catalyst washedwith hot water (60° C., 3 ml) and the combined filtrates concentrated invacuum. The residue was dissolved in 28% aqueous ammonium hydroxide (3ml), stirred at room temperature for 3 hours, concentrated in vacuum andpurified by column chromatography on DEAE cellulose using a gradient of0.05M-0.5M TEAB buffer. The product elutes at a buffer concentration of0.3-0.4M. The fractions containing the product were evaporated and usedfurther.

N⁶-(6-Tamra-amidohexyl)-2′-deoxyisoGuanosine 5′-triphosphate 8a

Compound 7 (approximately 1 mg as its triethylammonium salt) wasdissolved in 0.2 ml 0.1M TEAB buffer and 6-carboxytetramethylrhodamineN-hydroxysuccinimide ester (10 mg), dissolved in DMF (0.2 ml), wasadded. It was stirred at 35° C. for 3 hours, then omega-aminobutylagarose added to bind the excess Tamra™, stirred for another hour andthe reaction mixture loaded to a DEAE cellulose column, which was elutedwith a gradient of 0.05M-0.5M TEAB buffer. The product elutes at abuffer concentration of 0.4M. The fractions containing the product wereevaporated at 30° C.

Compounds 8b and 8d will be prepared in the same way as compound 8a,using 6-carboxyfluorescein N-hydroxysuccinimide ester and QSY7™N-hydroxysuccinimide ester, respectively, instead of6-carboxytetramethylrhodamine N-hydroxysuccinimide ester.

N⁶-(6-Biotinylamidohexyl)-2′-deoxyisoGuanosine 5′-triphosphate 8c

Compound 7 (approximately 1 mg as its triethylammonium salt) wasdissolved in 0.2 ml water and biotin 2-nitrophenyl ester (10 mg),dissolved in DMF (0.2 ml), was added. The solution turned to lightyellow. It was stirred at 35° C. for 1 hour, then omega-aminobutylagarose added to bind the excess biotin, stirred for another hour andthe reaction mixture loaded to a DEAE cellulose column, which was elutedwith a gradient of 0.05M-0.5M TEAB buffer. The product elutes at abuffer concentration of 0.4M. The fractions containing the product wereevaporated at 30° C.

Section B

For the following chemical reactions involving the synthesis oflabeled—deoxyisoGuanosine 5′-Triphosphates using Process A,tributylammonium pyrophosphate was purchased from Sigma; biotinN-hydroxysuccinimide ester, was purchased from Pierce Chemical Company;QSY7™ N-hydroxysuccinimide ester and Dabcyl N-hydroxysuccinimide werepurchased from Molecular Probes; all other chemicals were purchased fromAldrich Chemical Co. or Fisher Chemical Co. and were used withoutfurther purification. Solvents were dried over 4 Å molecular sieves.Reactions were carried out under dry argon in oven-dry glassware. Columnchromatography was performed with silica gel (230-425 mesh).

The following abbreviations were used: Ac₂O (Acetic anhydride); DMF(N,N-Dimethylformamide); DMAP (4,4′-Dimethylaminopyridine); DMT(4,4′-Dimethoxytrityl); Et₃N (Triethylamine); MeCN (Acetonitrile); MeOH(Methyl alcohol); Tol (p-Toluyl).

1-(p,p′-Dimethoxytrityl)-hexamethylenediamine (9)

Hexamethylenediamine (10 eq., 375 mmol, 43.5 g) was coevaporated twotimes from pyridine and dissolved in 100 ml pyridine. DMAP (0.1 eq.,3.75 mmol, 457 mg) was added and the reaction flask placed in an icebath. DMT-chloride (1 eq., 37.5 mmol, 12.69 g), dissolved in 100 mlpyridine, was added dropwise over 2 h. It was stirred at roomtemperature for 4 h, MeOH (5 ml) added, the reaction mixtureconcentrated and the remaining residue extracted with aqueousNaHCO₃/ethyl acetate. The organic layer was washed twice with aqueousNaHCO₃ solution, dried and the solvent evaporated. The obtained productwas used in next step without further purification.

Yield: 14.895 g (35.634 mmol, 95%) sticky oil.

2-Chloro-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxy-3′,5′-ditoluoylriboside(10)

Compound 9 (1.3 equiv., 31.916 mmol, 13.34 g) was coevaporated with DMFand dissolved in 100 ml DMF. Diisopropylethylamine (3.9 equiv., 95.748mmol, 16.65 ml) and compound 1 (1 equiv., 24.551 mmol, 13.282 g),dissolved in 100 ml DMF, were added and it was stirred at roomtemperature for 3 h. It was concentrated, the residue extracted withaqueous NaHCO₃/ethyl acetate, the organic layer dried and the solventevaporated. The residue was triturated with ether twice and the obtainedsolid product used further after drying in vacuum without furtherpurification.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxyriboside(11)

Compound 10 (1 equiv., 19.23 mmol, 17.74 g) was dissolved in DMF (25 ml)and added to a solution of NaH (10 eq., 192.3 mmol, 7.69 g of a 60%dispersion in mineral oil) in benzylalcohol (128 mL). The reactionmixture was heated (120° C., 6 h) and then stirred at room temperature(15 h) before filtrated over Celite™, the filtrate evaporated, theresidue extracted (ethyl acetate/water), the organic layer washed(NaHCO₃-solution), dried, the solvent evaporated and the residuetriturated 5 times with ether/hexane 1:10. TLC: CHCl₃/10% MeOHR_(F)=0.26.

Yield: 10.280 g (13.562 mmol, 70.5% for 2 steps) foam.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-2′-deoxy-5′-O-p,p′-dimethoxytritylriboside(12)

Compound 11 (14.7388 mmol, 11.172 g) was coevaporated with pyridine,dissolved in 150 ml pyridine and DMAP (0.25 equiv., 3.6847 mmol, 450 mg)added. The flask was placed in an ice bath and DMTC1 (1.5 equiv., 22.108mmol, 7.484 g) was added slowly over 2 h. It was stirred at roomtemperature for 22 h, then MeOH (1 ml) added, the reaction mixtureconcentrated and the residue extracted (chloroform/aqueous NaHCO₃). Theorganic layer was dried, the solvent evaporated and the residuetriturated with ether/hexane 1:1 to remove the excess DMT and theinsoluble solid product was dried and used further without additionalpurification.

Yield: 14.890 g (14.047 mmol, 95%) light brown foam.

2-Benzyloxy-6-(6-p,p′-dimethoxytritylaminohexyl)-aminopurine-3′-O-acetyl-2′-deoxy-5′-O-p,p′-dimethoxytritylriboside(13)

Compound 12 (14.047 mmol, 14.89 g) was coevaporated with pyridine,dissolved in 200 ml pyridine and DMAP (0.25 equiv., 3.5117 mmol, 428mg), Et₃N (5 equiv., 70.235 mmol, 9.7 ml) and Ac₂O (2.5 equiv., 35.1175mmol, 3.582 g) were added. It was stirred at room temperature for 4.5 h,then MeOH (2 ml) added, the reaction mixture concentrated and theresidue extracted (ethyl acetate/aqueous NaHCO₃). The organic layer wasdried, the solvent evaporated and the residue purified by columnchromatography using an one step gradient of ethyl acetate/hexane/Et₃N30:60:1, then 65:35:3. Yield: 5.93 g (5.385 mmol, 38%), yellow foam.

2-Benzyloxy-6-(6-aminohexyl)-aminopurine-3′-O-acetyl-2′-deoxyriboside(14)

Compound 13 (2.471 mmol, 2.723 g) was dissolved in 50 ml acetonitrile/2ml water and Ce(NH₄)₂(NO₃)₃ (0.3 equiv., 0.74 mmol, 406 mg) was added.It was refluxed for 45 min., then another 0.15 equiv. Ce(NH₄)₂(NO₃)₃(0.37 mmol, 205 mg) added and refluxing continued for 1 h. Then, it wasevaporated, the residue triturated with ether to remove the DMT, theinsoluble product dried and used further without additionalpurification.

2-Benzyloxy-6-(6-trifluoroacetamidohexyl)-aminopurine-3′-O-acetyl-2′-deoxyriboside(15)

The above obtained compound 14 (max. 5.385 mmol) was dissolved in 30 mlMeOH/50 ml ethyl trifluoroacetate/5 ml Et₃N and the reaction mixturestirred at room temperature for 21.5 h. TLC (chloroform/17.5% MeOH):R_(F)=0.72) indicated complete conversion. It was evaporated, theresidue extracted (brine/ethyl acetate), the organic layer dried, thesolvent evaporated and the residue purified by silica gel columnchromatography using a one step gradient of chloroform/1.5% MeOH, then17.5% MeOH. Yield: 2.80 g (4.714 mmol, 87%) foam.

2-Benzyloxy-6-(6-trifluoroacetamidohexyl)-aminopurine-3′-O-acetyl-5′-triphosphoryl-2′-deoxyriboside(16)

Imidazole (61 eq., 306 mg, 4.5 mmol, recrystallised) was dissolved inacetonitrile (3.6 mL) and chilled (0° C.). POCl₃ (19 eq., 0.128 mL) andtriethylamine (61 eq., 0.633 mL) were then added and the mixture wasstirred (0° C., 0.5 h) before adding a portion (0.309 mL) to 15 (1 eq.,0.074 mmol, 44 mg). This mixture was stirred (r.t., 0.5 h) before addingDMF (1.5 mL) containing tributylammonium pyrophosphate (2 eq., 0.16mmol, 73 mg). The reaction was then quenched (2 mL, 10% NH₄COO) 24 hlater and lyophillized. Product was purified by anion-exchangechromatography (Dionex ProPac™ SAX-10; Dionex, Sunnyvale, Calif.) using20% MeCN and a gradient of (NH₄)₂CO₃/20% MeCN. Collected product wasrepetitively lyophilized to remove excess salt. Yield 0.007 mmol (10%),white solid.

6-(6-aminohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside (7)

Compound 16 (0.007 mmol) was dissolved in methanol (2.5 mL) beforeadding Pd/C (10%, 5 mg) and NH₄COO (0.05 mmol, 31 mg). The suspensionwas refluxed (1 h) before filtering off the catalyst and evaporating thesolvent. The residue was then treated with 28% ammonium hydroxide (1.5mL, 3 h, room temp.) before the reaction was dried and the productpurified by anion-exchange chromatography (Dionex ProPac™ SAX-10;Dionex, Sunnyvale, Calif.) using 20% MeCN and a gradient of(NH₄)₂CO₃/20% MeCN. Collected product was repetitively lyophilized toremove excess salt. Yield 0.0063 mmol (90%), white solid.

6-(6-biotinylamidohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside(8c)6-(6-dabcylamidohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside(17a) 6-(6-QSY7™amidohexyl)-aminopurine-5′-triphosphoryl-2′-deoxyriboside (17b)

To 7 (0.88 μmol, triethylammonium salt) in H₂O (40 μL) was added sodiumborate (10.5 μL, 1M, pH 8.5) followed by DMF (216 mL) containing biotinN-hydroxysuccinimide ester, dabcyl N-hydroxysuccinimide ester, orQSY7™-N-hydroxysuccinimide ester (2.6 μmol, 3 eq.). The reactionproceeded (3 h, 55° C.) before it was diluted with 20% MeCN and theproduct purified by anion-exchange chromatography (Dionex ProPac™SAX-10; Dionex, Sunnyvale, Calif.) using 20% MeCN and a gradient of(NH₄)₂CO₃/20% MeCN. Yields 50-80%.

Example 8 “Real Time” Monitoring of PCR Amplification by Site SpecificIncorporation of a Fluorescence-Quenching Nonstandard Deoxy-NucleotideTriphosphate

Monitoring of the fluorescence of PCR reactions was performed during thecycling of the PCR reactions. PCR reactions included a first and secondprimer and the second primer contains a fluorophore-coupled nucleotide(FAM-dT) at its 5′ end. During amplification of the template nucleicacid, a standard nucleoside triphosphate (reaction A; dTTP) or an isoGnucleoside triphosphate (reaction B; dGisoTP) coupled to a fluorescencequenching compound (Dabcyl or QSY7™) is incorporated [opposite andadjacent] the fluorophore-coupled nucleotide (FAM-dT) of the secondprimer, reducing the fluorescence signal in the PCR reaction.

The following nucleic acids were used in PCR reactions for this example:

Nucleic Acid Component Sequence SEQ ID NO: First Primer5′-GTYATYTGCG-c3-TCGTGCGGTGCGTC-3′ SEQ ID NO: 18 Second3′-TGTGTCGTGTCGTCCGAT-FAM 5′ SEQ ID NO: 19 Primer A Second3′-TGTGTCGTGTCGTCCGXT-FAM 5′ SEQ ID NO: 20 Primer B Template5′-TCGTGCGGTGCGTCACACAGCACAGCAGGC-3′ SEQ ID NO: 21

“c3” indicates a propyl spacer which was chemically installed in placeof a nucleotide during synthesis of the first primer. Thephosphoramidite used in the synthesis of the first primer was3-O-Dimethyltrityl-propyl-1-[2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Spacer Phosphoramidite C3; Glen Research, Sterling, Va.). Optionally,an oligonucleotide containing an identical nucleotide sequence,including nucleotide modifications, but lacking the propyl spacer can beused as a substitute for the first primer in the PCR reaction.

The following components (base PCR reaction components) at the indicatedconcentrations were present in all PCR reactions:

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Bovine Serum Albumin 0.1 μg/μl Sigma, St. Louis, MOTween ™ 20 0.1% EM Sciences, Gibbstown, NJ d-Trehalose 37.5 mM Aldrich,Milwaukee, WI Potassium acetate 40 mM Sigma, St. Louis, MO MgCl₂ 3 mMSigma, St. Louis, MO Klen-Taq ™ DNA 0.1 units/μL AbPeptides, St. Louis,MO polymerase dATP 50 μM Promega, Madison, WI dGTP 50 μM Promega,Madison, WI dCTP 50 μM Promega, Madison, WI First Primer 0.2 μM Template0.4 pM

The components indicated above were prepared.

Second primer A, second primer B, dTTP, Dabcyl dTTP (Glen Research,Sterling, Va.), Dabcyl dGisoTP, and QSY7™ dGisoTP were variablecomponents in the following PCR reactions. These components were addedto PCR reactions A through J, as indicated below, at the indicatedconcentrations:

PCR Reaction: Variable PCR components (in addition to base components):A 0.2 μM Second Primer B, and 50 μM dTTP B 0.2 μM Second Primer A, and 1μM Dabcyl dTTP C 0.2 μM Second Primer B, and 1 μM Dabcyl dGisoTP D 0.2μM Second Primer B, and 50 μM dTTP E 0.2 μM Second Primer B, and 5 μMDabcyl dTTP F 0.2 μM Second Primer B, 50 μM dTTP, and 5 μM DabcyldGisoTP G 0.2 μM Second Primer B, 50 μM dTTP, and 5 μM QSY7 ™ dGisoTP H0.2 μM Second Primer A, and 50 μM dTTP I 0.2 μM Second Primer A, and 5μM Dabcyl dTTP J 0.2 μM Second Primer A, 50 μM dTTP, and 5 μM QSY7 ™dGisoTP

Reactions were prepared for a 25 μL final volume. Reactions mixtureswere loaded in 25 μL Smart Cycler PCR tubes (Cepheid, Sunnyvale,Calif.). The PCR tubes were spun in a mini-centrifuge for 6 seconds topull liquid into the reaction chamber. Tubes containing PCR reactionswere then placed in a Smart Cycler™ (Cepheid, Sunnyvale, Calif.) toprovide constant monitoring of fluorescence during the entire PCRreaction.

Thermal Cycling Parameters:

Cycle # Step Temp Time 1 1 95° C.  5 min. 2-41 1 95° C.  1 sec. 2 58° C. 1 sec.  3* 72° C. 10 sec. *During Step 3 of cycles 2-41 the optics ofthe Smart Cycler ™ were activated, allowing determination of thefluorescence in the PCR reaction tube.

Following the 41 cycles of PCR amplification, PCR reaction products weresubject to a melt curve analysis by increasing the temperature in thePCR tubes from 60° C. to 95° C. at a rate of 0.2° C. per second withfluorescence monitoring optics on.

Fluorescence quenching of PCR reaction products from PCR reactions A, B,and C is shown in FIG. 17A and the melting curve analysis of these PCRproducts is shown in FIG. 17A. These results demonstrate that thefluorescence of the PCR reaction is quenched in samples that include aquenching compound-coupled standard nucleoside triphosphate or aquenching compound-coupled isoG nucleoside triphosphate in combinationwith the a fluorophore-coupled standard nucleoside-containing secondprimer or nonstandard nucleoside-containing second primer, respectively.The melting curve data indicates that the fluorescence in the reactionproducts is restored by separating the fluorophore-coupled nucleic acidstrands from the quenching compound-coupled nucleic strands.

Fluorescence quenching of PCR reaction products from PCR reactions D, E,F, and G is shown in FIG. 18A Fluorescence quenching of PCR reactionproducts from PCR reactions H, I, and J is shown in FIG. 18B.

Example 9 Real Time Quantitation of Genomic DNA

Monitoring of the fluorescence of PCR reactions was performed during thecycling of the PCR reactions and amplification of nucleic acid templatefrom a genomic DNA sample. PCR reactions included a first primer andsecond primer containing two non-standard nucleotides (iso-G and iso-C);the primers were designed to hybridize and to amplify a region of mousegenomic DNA. PCR reactions also included a reporter nucleic acidcontaining a non-standard nucleotide (iso-G), a fluorescence quenchingcompound-coupled nucleotide (Dabcyl dT), and a fluorophore (6FAM)coupled to the 5′ base (T) of the reporter. Annealing of the reporternucleic acid to the amplification product, including pairing of thenon-standard nucleotides, can cause the cleavage of the fluorophore fromthe reporter nucleic acid containing the fluorescence quenchingcompound-coupled nucleotide by nucleic acid polymerase activity.

The following nucleic acids were used in PCR reactions for this example:

Nucleic acid component Sequence Seq ID# Reporter5′-FAM-TYQCCTGTCTGCCTGT-3′ SEQ ID NO: 22 First5′-GATAATCAGTAGCTTTGTAACCCTG-3′ SEQ ID NO: 23 Primer A First5′-GTGGCACAAGATTGATGGAAT-3′ SEQ ID NO: 24 Primer B Second3′-CATGTCATTTGTCAACCACCCYXAGGA- SEQ ID NO: 25 Primer A CAGACGGACAGCAC-5′Second 3′-CAATGACGTCGTTCCAGGAYXAGGAC- SEQ ID NO: 26 primer BAGACGGACA-5′ Template A Mouse genomic DNA; Strain: A/J (target  locus L11316, Chromosome 3 - 9.679 P) Template BMouse genomic DNA; Strain: C57BL/6J (target locus R75378, Chromosome 10 - 41.5 F)

Mouse genomic DNA was obtained from Jackson Laboratories (Bar Harbor,Me.) and diluted in 1 mM MOPS pH7.5, 0.01 mM EDTA. For template A, Mousestrain A/J genomic DNA was serially diluted to concentrations of 5ng/μl, 2.5 ng/μl, 1.25 ng/μl, 0.63 ng/μl, 0.31 ng/μl, and 0.16 ng/μl.For template B, the Mouse strain C57BL/6J genomic DNA was seriallydiluted to concentrations of 20 ng/μl, 2 ng/μl, 0.2 ng/μl, 20 pg/μl, 2pg/μl, and 0.2 pg/μl. The genomic DNA dilution series were boiled for 5min., placed on ice for 5 min., and stored at −20° C.

Primers were synthesized for the PCR amplification and detection ofspecific target nucleic acid sequences in the mouse genome. An initialtarget nucleic acid sequence was chosen to assess the viability of thisprocedure using A/J mouse genomic DNA, locus L11316, Chromosome 3—9.679P (design A). The target sequence chosen for further genomic DNAquantitation was mouse strain C57BL/6J, locus R75378, Chromosome 10—41.5F (design B). First primer A and first primer B are designed to have aTm between 60.0-63.0° C. Second primer A and second primer B aredesigned to have a Tm between 61.0-63.0° C. All primers were assessedfor secondary structure formation using Oligo 4.0™ Software forMacintosh (National Biosciences, Minneapolis, Minn.).

The following components (base PCR reaction components) at the indicatedconcentrations were present in all PCR reactions in this example:

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Bovine Serum Albumin 0.1 μg/μL Sigma, St. Louis, MOTween ™ 20 0.1% EM Sciences, Gibbstown, NJ d-Trehalose 37.5 mM Aldrich,Milwaukee, WI Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂ 2 mMSigma, St. Louis, MO Amplitaq ™ DNA 0.025 units/μL Applied Biosystems,Foster polymerase City, CA dATP 25 μM Promega, Madison, WI dGTP 25 μMPromega, Madison, WI dCTP 25 μM Promega, Madison, WI dTTP 25 μM Promega,Madison, WI First Primer (A or B) 0.2 μM Second Primer (A or B) 0.2 μMReporter 0.2 μM

Master mix A containing the ingredients listed above (with First PrimerA and Second Primer A) was prepared at a 1.04× concentration for 25 μLfinal reaction volumes.

Master Mix B containing the ingredients listed above (with First PrimerB and Second Primer B) was prepared at a 1.25× concentration for 25 μLfinal reaction volumes.

Design A reaction mixtures were created by adding 1 μL of each A/Jgenomic target DNA dilutions of 5 ng/μL, 2.5 ng/μL, 1.25 ng/μL, 0.63ng/μL, 0.31 ng/μL, and 0.16 ng/μL to 25 μL Smart Cycler™ PCR tubescontaining 24 μL of PCR A Master mix. Tubes were spun in amini-centrifuge for 6 seconds to pull liquid into the reaction chamber.Individual PCR tubes contained 5 ng, 2.5 ng, 1.25 ng, 630 pg, 30 pg, and160 pg of A/J genomic target DNA which corresponds to a nucleic acidtarget number of 1500, 750, 375, 188, 94, and 47 haploid equivalents,respectively.

Design B reaction mixtures were created by adding 5 μL of each C57BL/6Jgenomic target DNA dilutions of 20 ng/μL, 2 ng/μL, 0.2 ng/μL, 20 pg/μL,and 2 pg/μL to a thermocycling plate well or tube specific for each realtime thermocycler containing 20 μL of PCR Master Mix B. Individual PCRtubes or wells contained 100 ng, 10 ng, 1 ng, 100 pg, and 10 pg ofC57BL/6J genomic target DNA which corresponds to a nucleic acid targetnumber of 30,000 targets, 3,000 targets, 300 targets, 30 targets, and 3targets, respectively. When reactions were run in microtiter plates, a15 μL mineral oil overlay was added to each well prior to thermocyclingin order to prevent evaporation of the sample volume.

Design A reaction mixtures were placed into the Smart Cycler™ and cycledunder the following conditions:

Cycle # Step Temp Time 1 1 90° C. 30 min.  2-16 1 90° C.  1 sec. 2 56°C.  1 sec. 17-51 1 90° C.  1 sec.  2* 56° C. 11 sec. *During Step 2 ofcycle #17-51 the optics of the Smart Cycler ™ were activated, allowingdetermination of the fluorescence in the PCR reaction tube in order togenerate a kinetic plot.

The fluorescence reading for these reactions are shown in FIG. 19.

Design B reaction mixtures were individually placed into the followingreal-time PCR thermocyclers:

-   -   1) Smart Cycler™ (Cepheid; Sunnyvale, Calif.) using Smart        Cycler™ 25 μL Tubes (Cepheid; Sunnyvale, Calif.)    -   2) Light Cycler™ (Roche; Basel, Switzerland) using Light Cycler™        Tubes (Roche; Basel, Switzerland)    -   3) iCycler™ (BioRad; Hercules, Calif.) using 96-well Microtiter        Plates (MJ Research Inc.; Waltham, Mass.)    -   4) 7700 (Applied Biosystems; Foster City, Calif.) using        MicroAmp™ Optical 96-Well Reaction plate wells (Applied        Biosystems; Foster City, Calif.) and cycled under the following        conditions:

Cycle # Step Temp Time 1 1 95° C.  5 min. 2-46 1 95° C.  5 sec. 2 56° C.10 sec.  3* 60° C. 10 sec. *During Step 3 of cycle #2-46 the optics ofthe real-time PCR thermocyclers were activated to read FAM-generatedfluorescence, allowing determination of the fluorescence in the PCRreaction tube in order to generate a kinetic plot.

Fluorescence readouts were analyzed by the threshold cycle (Ct) andcorrelation of variance (Cv) method. The threshold cycle is when thesystem begins to detect the increase in the signal associated with anexponential growth of PCR product during the log-linear phase. The slopeof the log-linear phase is a reflection of the amplification efficiencyand bona fide amplification is indicated by an inflection point in theslope, the point on the growth curve when the log-linear phase begins.This point also represents the greatest rate of change along the growthcurve. Nucleic acid quantitation is correlated with the Ct wherein thegreater the initial amount of nucleic acid, the lower the Ct value. Ctshould be placed above any baseline activity and within the exponentialincrease phase.

Example 10 Real Time Quantitation of RNA

Monitoring of the fluorescence of PCR reactions was performed during thecycling of the PCR reactions and amplification of nucleic acid templatefrom a RNA sample. DNA primers were synthesized for the detection andquantitation of human β-actin mRNA. The cDNA/first primer hybridizes toa sequence on the 5′ region of human β-actin mRNA and primes cDNAsynthesis using reverse transcriptase. The cDNA/First primer and theSecond Primer, which contains two non-standard nucleotides (iso-C andiso-G), are then used for amplification of the human β-actin sequenceusing the cDNA as a template. PCR reactions also included a reporternucleic acid containing a non-standard nucleotide (iso-G), afluorescence quenching compound-coupled nucleotide (Dabcyl dT), and afluorophore (6FAM) coupled to the 5′ base (T) of the reporter. Annealingof the reporter nucleic acid to the amplification product, includingpairing of the non-standard nucleotides, can cause the cleavage of thefluorophore from the reporter nucleic acid coupled to the fluorescencequenching compound by nucleic acid polymerase activity.

The following nucleic acids were used in reverse transcription-PCR(RT-PCR) reactions for this example:

Nucleic acid component Sequence SEQ ID NO Reporter3′-TGTCCGTCTGTCCQYT-FAM-5′ SEQ ID NO: 27 cDNA/First3′-CTACTATAGCGGCGCG-5′ SEQ ID NO: 28 Primer Second Primer5′-CACGACAGGCAGACAGGAXYCGCCAG- SEQ ID NO: 29 CTCACCATG-3′ Templatehuman cardiac RNA (single donor)

Total human cardiac RNA from a single donor was obtained from Clontech(Palo Alto, Calif.).

RNA samples were diluted to 20 ng/μl, 2 ng/μl, 200 pg/μl, 20 pg/μl, 2pg/μl and 0.2 pg/μl in a buffer composed of 5 mM Bis-Tris-Propane pH8.9, 0.1 mM EDTA, 100 ng/ml yeast tRNA (Sigma, St. Louis, Mo.) and 100ng/ml sheared herring sperm DNA (Sigma, St. Louis, Mo.).

The following components (base PCR reaction components) at the indicatedconcentrations were present in all PCR reactions:

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Bovine Serum Albumin 0.1 μg/μL Sigma, St. Louis, MOTween ™ 20 0.1% EM Sciences, Gibbstown, NJ d-Trehalose 37.5 mM Aldrich,Milwaukee, WI Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂ 3 mMSigma, St. Louis, MO Ampli-taq ™ DNA 0.05 U/μL Applied Biosystems,Foster polymerase City, CA Reverse Transcriptase 0.05 U/μL Promega,Madison, WI (AMV) dATP 50 μM Promega, Madison, WI dGTP 50 μM Promega,Madison, WI dCTP 50 μM Promega, Madison, WI dTTP 50 μM Promega, Madison,WI cDNA/First Primer 0.2 μM Second Primer 0.2 μM Reporter 0.2 μM

An RT-PCR Master mix containing the reagents listed in Table X wasprepared at a 1.25× concentration for a 25 μL final reaction volumeusing nuclease-free H₂O. RT-PCR reactions mixtures were prepared byadding 20 μL of 1.25×RT-PCR Master mix to 5 μL of each diluted RNAsample in 25 μL Smart Cycler™ PCR tubes. Tubes were then spun in amini-centrifuge for 6 seconds to pull liquid into the reaction chamber.

Following centrifugation, reaction mixtures were placed immediately intothe Smart Cycler™ and cycled under the following conditions:

Cycle # Step Temp Time 1. 1 60° C.  1 min. 2. 1 95° C.  5 min. 3-52 194° C.  1 sec.  2* 60° C. 10 sec. *During Step 2 of cycle #3-52 theoptics of the real-time PCR thermocyclers were activated to readFAM-generated fluorescence, allowing determination of the fluorescencein the PCR reaction tube in order to generate a kinetic plot. Theresults are shown in FIG. 20.

Example 11 Real Time Quantitation of RNA by Site Specific Incorporationof Labeled Non-Standard Bases

Monitoring of the fluorescence of PCR reactions was performed during thecycling of the PCR reactions and amplification of nucleic acid templatefrom a RNA sample. DNA primers were synthesized for the detection andquantitation of human β-actin mRNA. The cDNA/first primer hybridized toa sequence on the 5′ region of human β-actin mRNA and primed cDNAsynthesis using reverse transcriptase. The cDNA/First primer and asecond primer containing a fluorophore (6FAM) coupled to the 5′ base (T)of the reporter and a 5′-penultimate non-standard nucleotide (iso-dC)were then used for amplification of the human β-actin sequence using thecDNA as a template. During amplification of the template nucleic acid, afluorescence quenching compound-coupled nonstandard nucleosidetriphosphate (Dabcyl-d-isoGTP) was present in the PCR reaction andincorporated opposite the nonstandard nucleotide (iso-dC) of the secondprimer, which is adjacent the fluorophore-coupled 5′-nucleotide(FAM-dT), and reduces the fluorescence signal in the PCR reaction.

The following nucleic acids were used in RT-PCR reactions for thisexample:

Nucleic acid component Sequence SEQ ID NO: cDNA/First3′-CTACTATAGCGGCGCG-5′ SEQ ID NO: 28 Primer Second 5′FAM-TXCGCCAGCTCACCATG-3′ SEQ ID NO: 30 Primer Templatehuman cardiac RNA (single donor)

Total human cardiac RNA from a single donor was obtained from Clontech(Palo Alto, Calif.)

RNA samples were diluted to 20 ng/μL, 2 ng/μL, 200 pg/μL, 20 pg/μL, 2pg/μL and 0.2 pg/μL in a buffer composed of 5 mM Bis-Tris-Propane pH8.9, 0.1 mM ETDA, 100 ng/mL yeast tRNA (Sigma, St. Louis, Mo.) and 100ng/mL sheared herring sperm DNA.

The following components (base PCR reaction components) at the indicatedconcentrations were present in all PCR reactions:

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Bovine Serum Albumin 0.1 ug/μL Sigma, St. Louis, MOTween ™ 20 0.1% EM Sciences, Gibbstown, NJ d-Trehalose 37.5 mM Aldrich,Milwaukee, WI Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂ 3 mMSigma, St. Louis, MO Klen-Taq ™ DNA 0.025 U/μL AbPeptides, St. Louis, MOpolymerase Reverse Transcriptase 0.5 U/μL Ambion, Austin, TXMaloney-Murine Lukemia Virus (M-MLV-RT) dATP 50 μM Promega, Madison, WIdGTP 50 μM Promega, Madison, WI dCTP 50 μM Promega, Madison, WI dTTP 50μM Promega, Madison, WI Dabcyl-d-isoGTP 2 μM Eragen Biosciences,Madison, WI cDNA/First Primer 0.2 μM Second Primer 0.2 μM

An RT-PCR Master mix was prepared for a 25 μL final reaction volume at a1.25× concentration using nuclease-free H₂O. RT-PCR reactions mixtureswere prepared by adding 20 μl of 1.25×RT-PCR Master mix to 5 μL of eachdiluted RNA sample in 25 μL Smart Cycler™ 25 μl Tubes (Cepheid,Sunnyvale, Calif.). Tubes were then spun in a mini-centrifuge for 6seconds to pull liquid into the reaction chamber.

Following centrifugation, reaction mixtures were placed immediately intoa Smart Cycler™ (Cepheid, Sunnyvale, Calif.), and cycled under thefollowing conditions:

Cycle # Step Temp Time 1. 1 60° C. 1 min. 2. 1 95° C. 5 min.  3-22 1 94°C. 1 sec. 2 60° C. 1 sec. 23-52 1 94° C. 1 sec. 2 60° C. 1 sec.  3* 72°C. 6 sec. *During Step 3 of cycle #23-52 the optics of the SmartCycler ™ are activated to read FAM-generated fluorescence, allowingdetermination of the fluorescence in the PCR reaction tube in order togenerate a kinetic plot as shown in FIG. 21.

Example 12 Multiplexed Allele Specific PCR

Multiplexed fluorescence-based PCR reactions were performed to determinethe sequence of a single nucleotide polymorphism mouse STS sequence27.MMHAP25FLA6 from various mouse strains. In this example, themultiplexed PCR reaction included a common first primer that hybridizesto a downstream non-polymorphic sequence on the target nucleic acid andtwo upstream second primers, second primer A and second primer B, eachsecond primer being allele-specific where the specificity was determinedby different 3′ nucleotides. Second primer A and second primer B alsohad different 5′ regions, which did not contribute to target nucleicacid hybridization but allow for hybridization of reporter A andreporter B, respectively. The reporter nucleic acids each contained a5′-penultimate non-standard nucleotide and a fluorescence quenchingcompound-coupled nucleotide and each contained 5′ nucleotides withdifferent fluorophores (either FAM or HEX) coupled to the 5′ nucleotide.The different fluorophores emitted different wavelengths of light uponexcitation. Annealing of the reporter nucleic acid to the amplificationproduct, including pairing of the non-standard nucleotides, can causethe cleavage of the fluorophore or fluorophores from the quenchingcompound-coupled reporter nucleic acid by nucleic acid polymeraseactivity. The predominance of an allele-specific nucleic acid targetresults in particular fluorescence emission of the fluorophore from thecleaved reporter.

The following nucleic acids were used in RT-PCR reactions for thisexample:

Nucleic acid component Sequence SEQ ID NO Reporter A5′-HEX-TYQGGACAGACG-3′ SEQ ID NO: 31 Reporter B 5′-FAM-TYQCCTGTCTGC-3′SEQ ID NO: 1 First Primer 3′-CAGTGACTGGCTGACGAG-5′ SEQ ID NO: 32 Second5′-CGTCTGTCCAXYGAGCTAGCGGAGGCC-3′ SEQ ID NO: 33 Primer A Second5′-GCAGACAGGAXYGGAGCTAGCGGAGGCT-3′ SEQ ID NO: 34 primer B Template 1Mouse genomic DNA; Strain: A/J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 2 Mouse genomic DNA; Strain: AKR/J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘TT’)Template 3 Mouse genomic DNA; Strain: BALB/cByJ ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 4 Mouse genomic DNA; Strain: C3H/HeJ ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 5 Mouse genomic DNA; Strain: C57BL/6J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘TT’)Template 6 Mouse genomic DNA; Strain: DBA/2J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 7 Mouse genomic DNA; Strain: AB6F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 8 Mouse genomic DNA; Strain: AKD2F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 9 Mouse genomic DNA; Strain: B6C3F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 10 Mouse genomic DNA; Strain: B6D2F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 11 Mouse genomic DNA; Strain: CByB6F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 12 Mouse genomic DNA; Strain: C3D2F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 13 Mouse genomic DNA; Strain: CByD2F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’) ±= inbred strains. ** = F1 hybrid strains.

Mouse gDNA samples were purchased from Jackson Laboratories (Bar Harbor,Me.). All gDNA samples were diluted to 2 ng/μL in 1 mM MOPS pH 7.5, 0.01mM EDTA.

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂2 mM Sigma, St. Louis, MO AmpliTaq ™ DNA 0.5 U/rxn Applied Biosystems,polymerase Foster City, CA dATP 50 μM Promega, Madison, WI dGTP 50 μMPromega, Madison, WI dCTP 50 μM Promega, Madison, WI dTTP 50 μM Promega,Madison, WI First Primer 0.2 μM Second Primer A 0.2 μM Second Primer B0.15 μM Reporter A 0.2 μM Reporter B 0.2 μM

A Master Mix containing components listed above was prepared at a 2×concentration for a final reaction volume of 10 μL. 5 μl of the MasterMix was aliquoted to individual wells of an assay plate and 5 μL oftarget DNAs (10 ng) were added to individual wells. PCR reactions wereprepared for positive controls (perfect match template) and negativecontrols (mismatch template or no template). After addition of templatenucleic acid each well was overlayed with 15 μL of mineral oil andcentrifuged briefly. Prior to running the PCR reaction, the assay platewas scanned for intensity of the fluorescent signal to establishbaseline fluorescence at 530 and 580 nm.

The following PCR parameters were used:

Cycle # Step Temp Time  1. 1 95° C.  5 min. 2-38. 1 95° C.  1 sec. 2 58°C.  1 sec. 39  1 49° C. 60 min. 40. 1  4° C. hold

Following PCR cycling reactions, the assay plates were tested foremissions of a fluorescence signal. The assay plates were transferred toCytofluor™ 4000 fluorescence plate reader (Applied Biosystems, FosterCity, Calif.) with the instrument set to read from the top of the plate.The parameters for the plate reader were as follows: (6FAM fluorescencedetection) excitation filter settings at 485±10 nm; emission filtersettings at 530±12.5 nm, and PMT gain set to 50, (HEX fluorescencedetection) excitation filter settings at 530±12.5 nm; emission filter to580±25 nm and PMT gain set to 50.

Example 13 Multiplex PCR Analysis of Factor V Genotype

Multiplexed fluorescence-based PCR reactions were performed to determineallele-specific nucleotide variations in the Factor V gene of humangenomic DNA. The procedure used in this example is similar to theprocedure used in Example 12.

Nucleic acid component Sequence SEQ ID NO Reporter5′-HEX-TYQGGACAGACG-3′ SEQ ID NO: 31 A Reporter 5′-FAM-TYQCCTGTCTGC-3′SEQ ID NO: 1 B First 5′-ATTTCTGAAAGGTTACTTCAAGGACA-3′ SEQ ID NO: 35Primer Second 3′-ACGGACAGGTCCCTAGAYXACCTGTCTGCCTGT-5′ SEQ ID NO: 36Primer A Second 3′-GCGGACAGGTCCCTAGYXAGGACAGACGGACA-5′ SEQ ID NO: 37primer B Template Synthetic Factor V wild type target; SEQ ID NO: 38 15′-ATTTCTGAAAGGTTACTTCAAGGACAAAATACCTG TATTCCTCGCCTGTCCAGGGATCTGCTCTTACAGA- 3′ TemplateSynthetic Factor V mutant target; SEQ ID NO: 39 25′-ATTTCTGAAAGGTTACTTCAAGGACAAAATACCTGTATTCCTTGCCTGTCCAGGGATCTGCTCTTACAGA- 3′ TemplateHuman genomic DNA including Factor V wild type target 3 TemplateHuman genomic DNA including Factor V mutant target 4

Synthetic Factor V targets were prepared by automated DNA synthesis.Human genomic DNA including Factor V targets was obtained from theCornell/NIGMS Human Genetic Cell Repository (Camden, N.J.). All gDNAsamples were diluted to 1 or 5 ng/μL in 1 mM MOPS pH 7.5, 0.1 mM EDTAand boiled 5 min then placed on ice prior to PCR. Synthetic targets wereserially diluted to 1 or 10 fM in 1 mM Tris pH 8.0 (Fisher Scientific,Pittsburgh, Pa.) and 0.1 μg/mL Herring Sperm DNA (St. Louis, Mo.).

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂2.5 mM Sigma, St. Louis, MO AmpliTaq ™ DNA 0.5 U/rxn Applied Biosystems,Foster City, polymerase CA dATP 50 μM Promega, Madison, WI dGTP 50 μMPromega, Madison, WI dCTP 50 μM Promega, Madison, WI dTTP 50 μM Promega,Madison, WI First Primer 0.4 μM Second Primer A 0.4 μM Second Primer B0.2 μM Reporter A 0.4 μM Reporter B 0.4 μM

A Master Mix containing components listed above was prepared at a 2×concentration for a final reaction volume of 10 μL. 5 μl of the MasterMix was aliquoted to individual wells of an assay plate (Low ProfileMultiplate™, 96 well; MJ Research, Waltham, Mass.) and 5 μl of targetDNAs were added to individual wells. 5 or 50 zmol (approximately 3000 or30,000 molecules) of mutant, wild type, or heterozygous synthetictargets were added to the wells. 5 or 25 ng of heterozygous, or wildtype human genomic DNA were added to the wells. Wells containing notarget DNA were used as controls. After addition of template nucleicacid each well was overlayed with 15 μl of mineral oil and centrifugedbriefly. Prior to running the PCR reaction, the assay plate was scannedfor intensity of the fluorescent signal to establish baselinefluorescence at 530 nm and 580 nm.

The following PCR parameters were used:

Cycle # Step Temp Time   1. 1 95° C.  5 min. 2-38. 1 95° C.  1 sec. 258° C.  1 sec. 39 1 49° C. 60 min.

Following PCR cycling reactions, the assay plates were tested foremissions of a fluorescence signal. The assay plates were transferred toCytofluor™ 4000 fluorescence plate reader (Applied Biosystems, FosterCity, Calif.) with the instrument set to read from the top of the plate.The parameters for the plate reader were as follows: (6FAM fluorescencedetection) excitation filter settings at 485±10 nm; emission filtersettings at 530±12.5 nm, and PMT gain set to 50, (HEX fluorescencedetection) excitation filter settings at 530±12.5 nm; emission filter to580±25 nm and PMT gain set to 50. The relative fluorescence units (RFUs)for HEX and FAM fluorescence, shown on the Y and X axes, respectively,for each multiplex PCR reaction performed are combined and shown in FIG.22.

Example 14 Multiplexed Real Time Allele Specific PCR Using ExonucleaseDeficient Nucleic Acid Polymerase and a Flap Endonuclease as a CleavingAgent

Multiplexed fluorescence-based PCR reactions were performed to thesequence of a single nucleotide polymorphism in the mouse STS sequence27.MMHAP25FLA6 of genomic DNA from various mouse strains. In thisexample, the multiplexed PCR reaction included a common first primerthat hybridized to a downstream non-polymorphic sequence on the targetnucleic acid and two upstream second primers, second primer A and secondprimer B, each second primer being allele-specific where the specificitywas determined by different 3′ nucleotides. Second primer A and secondprimer B also had different 5′ regions, which did not contribute totarget nucleic acid hybridization but allowed for hybridization ofreporter A and reporter B, respectively. The reporter nucleic acids eachcontained a 5′-penultimate non-standard nucleotide and a fluorescencequenching compound-coupled nucleotide but contained 5′ nucleotides withdifferent fluorophores (either FAM or HEX) coupled to the 5′ nucleotide.The different fluorophores emitted different wavelengths of light uponexcitation. Annealing of the reporter nucleic acid to the amplificationproduct, including pairing of the non-standard nucleotides, can causethe cleavage of the fluorophore or fluorophores from the quenchingcompound-coupled reporter nucleic acid by flap endonuclease-1 (FEN-1)enzyme activity. The predominance of an allele-specific nucleic acidtarget will result in particular fluorescence emission of thefluorophore from the cleaved reporter.

The following nucleic acids were used in RT-PCR reactions for thisexample:

Nucleic acid component Sequence SEQ ID NO Reporter A5′-HEX-TYQGGACAGACGGACA-3′ SEQ ID NO: 31 Reporter B5′-FAM-TYQCCTGTCTGCCTGT-3′ SEQ ID NO: 1 First Primer3′-CAGTGACTGGCTGACGAG-5′ SEQ ID NO: 32 Second5′TGTCCGTCTGTCCAXYGAGCTAGCGGAGG SEQ ID NO: 40 Primer A CC-3′ Second5′ACAGGCAGACAGGAXYGGAGCTAGCGGA SEQ ID NO: 41 primer B GGCT-3′ Template 1Mouse genomic DNA; Strain: A/J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CC’)Template 2 Mouse genomic DNA; Strain: AB6F1** (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘CT’)Template 3 Mouse genomic DNA; Strain: C57BL/6J ± (target:27.MMHAP25FLA6.seq located on mouse chomosome 2: seq. variation: ‘TT’) ±= inbred strains. ** = F1 hybrid strains.

Mouse gDNA samples were purchased from Jackson Laboratories (Bar Harbor,Me.). All gDNA samples were diluted to 20 ng/μL in 1 mM MOPS pH 7.5,0.01 mM EDTA and heated to 95 degrees C. for 5 minutes and snap cooledon ice.

Component 1X Conc. Supplier and location Bis-Tris-Propane pH 8.9 10 mMSigma, St. Louis, MO Potassium acetate 40 mM Sigma, St. Louis, MO MgC1₂2 mM Sigma, St. Louis, MO dATP 50 μM Promega, Madison, WI dGTP 50 μMPromega, Madison, WI dCTP 50 μM Promega, Madison, WI dTTP 50 μM Promega,Madison, WI First Primer 0.2 μM Second Primer A 0.2 μM Second Primer B0.15 μM Reporter A 0.2 μM Reporter B 0.2 μM Mja FEN-1 25.1 fmol/rxnMethanococcus jannaschii Platinum ™ GenoTYPE ™ 1.0 U/rxn LifeTechnologies, Tsp DNA Polymerase Rockville, MD

Mja FEN-1 was expressed and purified according to the method describedin Hosfield et al., J Biol Chem, 273:27154-61 (1998), hereinincorporated by reference, with modifications. The GenBank accessionnumber containing the Mja FEN-1 sequence is U67585. Mja FEN-1 isdescribed in U.S. Pat. No. 5,843,669, and Bult et al., Science,273:1058-1073 (1996), both herein incorporated by reference. The plasmidcontaining the Methanococcus jannaschii FEN-1 genes was transformed intothe E. coli strain BL21 (DE3) (Novagen, Madison, Wis.), and proteinoverexpression was induced in log phase by addition ofisopropylthiogalactopyranoside (Sigma, St. Louis, Mo.) to a finalconcentration of 0.4 mM. Following growth for an additional 2 hours thecells were pelleted at 3000×g, resuspended in Buffer 1 (10 mM Tris,pH7.5 (Fisher Scientific, Pittsburgh, Pa.), 150 mM NaCl (Sigma, St.Louis, Mo.), 10 mM Imidazole (Aldrich, Milwaukee, Wis.)), sonicatedbriefly, and lysed by heating at 75° C. for 45 minutes, and then cooledrapidly to 0° C. on ice. This protocol lysed the cells and precipitatedthe majority of the contaminating mesophilic native E. coli proteins.The resulting solution was centrifuged at 25,000×g, and the supernatantwas associated with TALON™ Metal Affinity Resin (Clontech, Palo Alto,Calif.) pre-equilibrated with Buffer 1, loaded into a gravity flowcolumn, and washed extensively with Buffer 1. FEN-1 was then elutedusing Buffer 1 adjusted to contain a stepwise imidazole gradient of 100mM, 200 mM, 350 mM and 500 mM. FEN-1 containing fractions were collectedand dialyzed extensively against a buffer containing 10 mM Tris, pH 7.5(Fisher Scientific, Pittsburgh, Pa.), 150 mM KCl, and 1 mM EDTA.Dialyzed material was adjusted to 50% glycerol (Fisher Scientific,Pittsburgh, Pa.), 0.5% Tween®20 (EM Sciences, Gibbstown, N.J.), and 0.5%Nonidet™ P-40 (Roche, Indianapolis, Ind.).

A Master Mix containing components listed above was prepared to be 1.5×the above final concentrations. 15 μl of these mixes were aliquoted toindividual wells of an assay plate and 5 μL of target DNAs (100 ng) wereadded to individual wells and mixed by aspiration. PCR reactions wereprepared for positive controls (perfect match template), negativecontrols (mismatch template or no template), and heterozygous sample(match and mismatch template). After template nucleic acid addition,each well was overlaid with 20 μL of mineral oil and centrifugedbriefly.

The assay plates were transferred to the iCycler iQ Real Time PCRDetection System (BioRad, Hercules, Calif.) and cycled using theparameters listed above. The filter sets used for signal detectionincluded: (6FAM)-excitation filter 490±10 nm, emission filter 530±15 nm;(HEX) excitation filter 530±15 nm, emission filter 575±10 nm.

The following PCR parameters were used:

Cycle # Step Temp Time   1. 1 95° C. 3 min.   2-26. 1 95° C. 1 sec. 259° C. 1 sec. 27-41 1 95° C. 1 sec.  2* 59° C. 2 min. 42 1  4° C. hold*During Step 2 of cycle #27-42 the optics of the iCycler iQ ™ Real TimePCR Detection System were activated to read FAM and HEX generatedfluorescence, allowing determination of the fluorescence in the PCRreaction tube in order to generate a kinetic plot. The results are shownin FIG. 23A-B.

Example 15 Melt Curve Analysis of Fluorescence-Quenched PCRAmplification Products and Fluorescence-Quenched PCR AmplificationProducts

Melting curve analysis of PCR reaction products containing fluorophoresquenched by SSI with a quenching compound can be used to examine thepresence of quencher-incorporated primer/dimers. Quencher-incorporatedprimer/dimers typically melt at temperature lower thanquencher-incorporated PCR products. Melt curve analysis can showfluorescence increases at the melting point of the quencher-incorporatedprimer/dimers and quencher-incorporated PCR products if both are presentas products following PCR amplification.

PCR amplification products from Example 11 were subjected to melt curveanalysis using the Smart Cycler™ (Cepheid, Sunnyvale, Calif.). Thechange in fluorescence was monitored while gradually increasing thetemperature of the PCR reaction products at a rate of 0.1° C. persecond. The Tm of the intended product (quencher-incorporated PCRproduct) as well as that of the nonspecific product(quencher-incorporated primer/dimers) is illustrated in FIG. 25. Themelt analysis for a RT-PCR reaction containing a starting quantity of 1pg of RNA template showed a significant amount of nonspecific productwith a Tm of approximately 71° C. as well as an intended product Tm ofapproximately 79° C. The melt analysis for a reaction containing 100 ngof RNA template showed only the formation the intended product with a Tmof 79° C. Once the Tm of the intended product is known it is conceivablethat in order to specifically observe the signal generated by theintended product by taking the fluorescent measurement of the reactionat a temperature above the Tm of the nonspecific product, it may beuseful to observe, and below that of the intended product. The resultsfrom the melt curve analysis are shown in FIG. 24.

What is claimed is:
 1. A kit for detecting a target nucleic acidcomprising: a) a first oligonucleotide primer comprising a sequencecomplementary to a first portion of the target nucleic acid; b) a secondoligonucleotide primer comprising a first region and a second region,the first region comprising a sequence complementary to a second portionof the target nucleic acid and the second region comprising anon-natural base; and c) a reporter comprising a label and a singlenon-natural base that is complementary to the non-natural base of thesecond region, wherein the reporter does not include any base other thanthe single non-natural base.
 2. The kit of claim 1, wherein the secondregion of the second oligonucleotide primer further comprises a labeland the labels of the reporter and the second region of the secondoligonucleotide primer comprise a pair of fluorophores where theemission of one of the fluorophores stimulates the emission of the otherfluorophore.
 3. The kit of claim 1, wherein the second region of thesecond oligonucleotide primer further comprises a label and the labelsof the reporter and the second region of the second oligonucleotideprimer comprise a signal generating element and a signal quenchingelement.